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Warren's Content

There have been 62 items by Warren (Search limited from 20-November 18)



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#64332 Nde1-BamH1 makes me sad

Posted by Warren on 26 March 2010 - 08:08 AM in Molecular Biology

I am curious about the NdeI -- awhile back in the middle of doing some more difficult cloning, I went ahead and made a deletion clone of a construct I had using NdeI, which should be the absolute easiest cloning you could possibly do, I was cutting out a chunk of DNA using just NdeI and religating (there was two sites in the insert). The digest looked perfect on a gel, and gave the expected two bands, and I gel purified the larger fragment and religated it -- literally about as simple as you can get as far as cloning -- and the first surprise was very few transformed colonies (should get TONS doing this) and second surprise was of the colonies I did get, none were correct, some strange plasmids (not the original clone either). I tried this again, and was extra careful and the same result -- since this was not anything critical I never spent time trying to figure out what was wrong, but I have done alot of cloning and never saw something like that with something seemingly so "easy". It makes me wonder about 1) using NdeI for cloning or 2) maybe some bizarre problem with the 2 bp AT overhangs and gel purification. Obviously I did try using lowered ligation temps (and using PEG which is already in the ligation buffer I use). But there is something weird there, and like someone already mentioned I am paranoid about using NdeI for cloning (although I use it for restriction digestions for sizing and it works fine).

Warren..


Hmm... looks like you aren't the only one being paranoid about Nde1.

I have BamH1(HF) in my lab and it cuts pretty well. I thought about switching BamH1(HF) to Xho1 and just use Nde1 and Xho1 for my digestion. But I am wondering if I should stick to BamH1(HF) as other people in my lab have told me that BamH1(HF) cuts pretty well and change Nde1 to another enzyme.

I have always had good luck with BamHI, and the HF version I suspect should be even better. Never had a problem with XhoI either that I can recall, just NdeI -- it may be due to something else but it had made me paranoid about trying it in the future, as of now I wouldn't knowingly use it to design a cloning strategy, but good luck anyways, can you use something other than NdeI? If it were me I and doing something different was not a real option I might even try blunting the NdeI site(s) and have BamHI at one end and a blunt end at the other, which I have done before and worked very well (the blunt end was via SmaI though).

Warren..


I do want to use BamH1(HF) too, for it works really well. But looking at the vector map, I will have to stick to using Nde1 as it will affect my gene orientation if I were to switch Nde1 to something else. :)


Actually, if you are using PCR to amplify the fragment you are cloning, you can still use BamHI and just leave the other end blunted. So, you can just phosphorylate the opposite PCR primer (near your NdeI site) and digest the PCR product with BamHI and clone it that way (you'll have to blunt the vector too, for example baqsed on your vector, cutting with XbaI and blunting it, or you can PCR amplify the vector as well). Having one sticky end and one blunt end is actually still pretty easy to clone, because the correct clone is still heavily favored in the reaction. Done it many times and you'll get fewer colonies than you would with two different sticky ends but you still should get plenty and most of them should be what you want (very low background). Sometimes you have to get creative like this when you run into these problems (many times its easier to just order new primers, all things considered!) Warren..



#63861 Nde1-BamH1 makes me sad

Posted by Warren on 24 March 2010 - 04:41 AM in Molecular Biology

I am curious about the NdeI -- awhile back in the middle of doing some more difficult cloning, I went ahead and made a deletion clone of a construct I had using NdeI, which should be the absolute easiest cloning you could possibly do, I was cutting out a chunk of DNA using just NdeI and religating (there was two sites in the insert). The digest looked perfect on a gel, and gave the expected two bands, and I gel purified the larger fragment and religated it -- literally about as simple as you can get as far as cloning -- and the first surprise was very few transformed colonies (should get TONS doing this) and second surprise was of the colonies I did get, none were correct, some strange plasmids (not the original clone either). I tried this again, and was extra careful and the same result -- since this was not anything critical I never spent time trying to figure out what was wrong, but I have done alot of cloning and never saw something like that with something seemingly so "easy". It makes me wonder about 1) using NdeI for cloning or 2) maybe some bizarre problem with the 2 bp AT overhangs and gel purification. Obviously I did try using lowered ligation temps (and using PEG which is already in the ligation buffer I use). But there is something weird there, and like someone already mentioned I am paranoid about using NdeI for cloning (although I use it for restriction digestions for sizing and it works fine).

Warren..


Hmm... looks like you aren't the only one being paranoid about Nde1.

I have BamH1(HF) in my lab and it cuts pretty well. I thought about switching BamH1(HF) to Xho1 and just use Nde1 and Xho1 for my digestion. But I am wondering if I should stick to BamH1(HF) as other people in my lab have told me that BamH1(HF) cuts pretty well and change Nde1 to another enzyme.

I have always had good luck with BamHI, and the HF version I suspect should be even better. Never had a problem with XhoI either that I can recall, just NdeI -- it may be due to something else but it had made me paranoid about trying it in the future, as of now I wouldn't knowingly use it to design a cloning strategy, but good luck anyways, can you use something other than NdeI? If it were me I and doing something different was not a real option I might even try blunting the NdeI site(s) and have BamHI at one end and a blunt end at the other, which I have done before and worked very well (the blunt end was via SmaI though).

Warren..



#63527 Nde1-BamH1 makes me sad

Posted by Warren on 22 March 2010 - 07:52 AM in Molecular Biology

I am curious about the NdeI -- awhile back in the middle of doing some more difficult cloning, I went ahead and made a deletion clone of a construct I had using NdeI, which should be the absolute easiest cloning you could possibly do, I was cutting out a chunk of DNA using just NdeI and religating (there was two sites in the insert). The digest looked perfect on a gel, and gave the expected two bands, and I gel purified the larger fragment and religated it -- literally about as simple as you can get as far as cloning -- and the first surprise was very few transformed colonies (should get TONS doing this) and second surprise was of the colonies I did get, none were correct, some strange plasmids (not the original clone either). I tried this again, and was extra careful and the same result -- since this was not anything critical I never spent time trying to figure out what was wrong, but I have done alot of cloning and never saw something like that with something seemingly so "easy". It makes me wonder about 1) using NdeI for cloning or 2) maybe some bizarre problem with the 2 bp AT overhangs and gel purification. Obviously I did try using lowered ligation temps (and using PEG which is already in the ligation buffer I use). But there is something weird there, and like someone already mentioned I am paranoid about using NdeI for cloning (although I use it for restriction digestions for sizing and it works fine).

Warren..



#58987 Strange Sequencing results

Posted by Warren on 15 February 2010 - 07:17 PM in Molecular Biology

Yes, this looks exactly as if two locations in the genome both amplified and differed in that region.


many thanks!

i was not sure how to deal with that ...but there must be something going within that spot.

Is there a way to dissect such trace overlays ...or do i have to do it manually?

Regards,
p


Why not just clone the PCR fragment and pick a few clones to get both sequences? Should be straight forward. Plus it will verify that you actually are seeing amplification of two different regions differing in those areas, and not some other strange artifact. It may turn out to something very cool.

Warren..



#58544 Reversible block, 3' OH?

Posted by Warren on 11 February 2010 - 10:54 AM in Molecular Biology

Suppose the following, I wish to ligate an ssRNA to the 3' end of a ssDNA. T4 RNA ligase should be able to do this. The ssDNA will not be phophorylated, so no issues there. The RNA will have a 5' phosphate. Now, my question is, is there a way to block the 3' OH to prevent self ligation or multiple ligations, ie, to make the desired hybrid the only possible ligation reaction? Importantly, this would need to be REVERSIBLE, ie, after the ligation I can remove the blocking group to yield a free 3' OH on the RNA. I know you can add dideoxy terminators and such, or chemically react something with the OH group, but once again I want to easily reverse this after the ligation to have a natural 3' OH group (and a specific one at that, so I don't want to add any modified nucleotides to the end). Any ideas? I haven't been able to find much online, but I may be searching with the wrong keywords.....warren...


Hi
I think You should consult the company/es that produce oligos like IDT.
They have different kinds of modifications for both 3' and 5' groups.
Modifications like amino blockers are widely used to prevent unwanted ligations but I'm not sure it is reversible.
Hope it is helpful.
Best
Michael


Thats not a bad idea.....



#58486 Reversible block, 3' OH?

Posted by Warren on 10 February 2010 - 09:56 PM in Molecular Biology

Suppose the following, I wish to ligate an ssRNA to the 3' end of a ssDNA. T4 RNA ligase should be able to do this. The ssDNA will not be phophorylated, so no issues there. The RNA will have a 5' phosphate. Now, my question is, is there a way to block the 3' OH to prevent self ligation or multiple ligations, ie, to make the desired hybrid the only possible ligation reaction? Importantly, this would need to be REVERSIBLE, ie, after the ligation I can remove the blocking group to yield a free 3' OH on the RNA. I know you can add dideoxy terminators and such, or chemically react something with the OH group, but once again I want to easily reverse this after the ligation to have a natural 3' OH group (and a specific one at that, so I don't want to add any modified nucleotides to the end). Any ideas? I haven't been able to find much online, but I may be searching with the wrong keywords.....warren...



#48626 Help needed for ligation of large inserts into TOPO vector

Posted by Warren on 30 November 2009 - 11:29 PM in Molecular Cloning

There are special ligases available for cloning large fragments, e.g. from Takara (www.takara-bio.us/files/fliyers/d473bbedc8f4d7defd12568a54fd7961.pdf). Normally, cloning of such a fragment should be no problem ...try different ratios of vector/insert as well as different ligation temperatures (RT, 16C, 4C) ...and check the ligation on a gel if you get any ligation products ...maybe your restriction enzymes are not cutting well (often the main problem!).

Regards,
p



yeah right, more often than not, its the inadequate digestion by RE's that leads to such problems.....i hv never had to clone these big inserts, but i hv seen some of my labmates doing it with normal ligases....So, make sure that REs have really worked well or not....
best.


This is a TOPO vector, so it doesn't use the traditional ligase, the ligase activity comes from the topoisomerase, and it is used to clone PCR products (not restriction fragments). I have not tried to use TOPO vectors recently to clone such large fragments, but I think it should work, if I remember right we had some clones somewhere in between what you are trying to make. One thing I have noticed is the efficiency seems to drop off alot as the product gets bigger, so your results with a 200bp fragment are probably not applicable. Then again, since those vectors are so expensive, I don't set up different insert ratios, since I can always get what I want, and the difference between getting 20 colonies and 500 doesn't matter! Anyway, we would need more details as to what you are doing in terms of PCR, purification, etc etc. My initial suggestion would be do a lot longer ligation time (rather than 5 minutes) maybe 1/2 to 1 hour, and do everything recommended for "maximum efficiency" in the manual, in terms of transformation and recovery. Any other recommendations would depend on what your current protocol is....warren..



#45740 3 way ligation help needed

Posted by Warren on 17 November 2009 - 07:30 PM in Molecular Cloning

I tried to ligate with the middle piece being CIP treated yesterday using (I had done all the other steps you suggested in the later post already and had the dna cut and cleaned up). I figured that since I only saw bands corresponding to the starting material that the ligase was bad so I acquired some new ligase and religated 100 ng of each of the 3 pieces (all similar size so 1:1:1 molar ratio) at room temperature for 1 hour (I have heard that NotI likes room temp ligation due to the high GC overhang). After running the ligation on a gel. I see bands at 700 and 720ish (which are my starting materials for the 1st and 3rd pieces) and a band at ~1400 (for any of the two pieces ligated) but no bands at 2000 plus.

I am now thinking I might have to put one piece into a cloning vector and purify and then use this cloning vector to put in the 2nd piece and so on. I really don't want to since it is time consuming but this 3 way ligation is killing me. I may also try ligating 2 of the pieces together and then PCRing and then trying to ligate this fragment with the 3rd piece.


Piecing it out might be easier in the long run -- sometimes easier techniques that appear to be more work or take longer, actually end up working quicker, I know this from experience!

However, if you were getting ladders before and now with a dephosphorylated middle fragment you are only getting your starting product only and single ligations, that is strongly suggestive that one of your restriction sites is not being cut, and based on your results so far (if I am understanding them correctly) it would most likely be one of your outer fragments. If you have some left over, you could check this quickly (I am assuming these are PCR products and will not ligate unless cleaved) -- just do a couple quick ligations with each of your outer fragments alone, which if they are cut correctly should yield a strong "double" band of ~1400 and a smaller band of ~700. The problem here is if one of your enzymes isn't cutting, doing it a longer way as you have suggested will not be any easier! How close to the end are your XhoI and NotI sites?

Another thing you can also do, since you mentioned the strong GC overhang, is briefly warm the sample to say 37 or 42 (not enough to denature the DNA, but enough to separate 4bp overhangs temporarily) will all your fragments are together right before you add the ligase (cool it back to room temp before adding the ligase). I do this sometimes, I don't know how much it helps, but I know it doesn't hurt.

Warren..



#45404 Creating mutants

Posted by Warren on 16 November 2009 - 10:35 PM in Molecular Cloning

I have to create some double mutants to see how they function. I have created the single mutants in these combinations and cloned them to a plasmid. The size of the plasmid is ~11kb. In order to create the double mutants I would like to get some advice as to the best possible way to do it with minimal sequencing, and if possible eliminate the cloning process, (which I guess would not be easy!)


Well, more details would be needed, but in an IDEAL situation, your single mutants would have a unique restriction site in between them, and one on one side or the other, and the double mutants could be created by simply switching fragments....it would require cloning obviously, but it would be extremely easy and no sequencing would be required (assuming your single mutants are already sequenced). However, more details are needed. Warren..



#45397 3 way ligation help needed

Posted by Warren on 16 November 2009 - 09:39 PM in Molecular Cloning

I tried it and it didn't work. I am wondering if I let the CIP reaction go to long. I let it sit for 10 min to cut 150 ng of DNA and the phosphate was removed on the 3'-5' strand.


ummm, ok, there is pretty much no way you could have done everything in the 2 hours since your last post, so I am wondering how you can know "it didn't work"? I guess I need to explain in more detail. I will operate under the assumption that your three fragments are PCR products (or at least the outside fragments, so your "Gateway sites" are not phosphorylated to begin with, ie they were part of the primer sequence of the original PCR). So, after the original PCRs, you do a PCR cleanup (ie column) or a gel purification. Then you digest your 3 fragments separately with the appropriate enzymes. Then you would take the middle fragment only, and dephosphorylate it. Then you would inactivate the phosphatase, and repurify your three digested fragments (to get rid of the small overhangs you cut off)...then add all three in equimolar amounts to a ligation reaction. In the ligation, everything that can happen will happen, so you will end up with bands at 700 (single fragments, may not be visible), 1400 (end fragments self-ligating) and 2100 (what you want, ie, the three correct fragments together). Nothing else should be possible if everything works (and your blunt gateway end can't ligate, ie, from a PCR). Then you would isolate the 2100 bp fragment from the gel and either clone it, or do PCR directly on it. You need to do this last purification as well, because if you don't, your 1400 bp fragments, which will be NotI or XhoI ends ligated together, will be able to amplify using just one of the outside primers, and would cause big problems in the PCR.

If you draw it out on a piece of paper, it might be easier to visualize. But it should work. The only problem would be doing PCR on nicked fragments, but there is enough overlap that you should get what you want.

warren..



#45313 3 way ligation help needed

Posted by Warren on 16 November 2009 - 12:49 PM in Molecular Cloning

Would that leave a system where there are no phosphates?

NotI leave the phosphate on the 5' end of my cut first fragement would have:

5'-Seq-GC-hydroxyl-3'

and my middle (xhoI and Not I would have)

5'-phosphate-GGCC (notI)-sequence-XhoI seq-hydroxyl-3'

and if I use CIP then it would remove the 5' phosphate so I'd be attempting to ligate a 3' end that has a hydroxyl from the first piece to the CIP treated 2nd piece with the 5' hydroxyl removed. I am assuming this would lead to no ligation.


DNA is double-stranded, so for instance in your first example you are showing only one of the strands, the other strand (with the 5' overhang) WILL be phosphorylated:

5'-Seq-GC-OH-3'
3'-Seq-CGCCGG-phos-5'

If you just dephosphoryate the middle fragment, the three fragments will ligate together the way you want, with a couple of nicks -- I'd probably try to clone it first before doing PCR. If your Gateway sites are blunt ends they can be ligated as well, but the overhangs will ligate preferentially.



#44864 Miniprep

Posted by Warren on 14 November 2009 - 06:33 PM in Molecular Cloning

AFAIK, it also depends on copy number of your plasmid in said bacteria. If your plasmid is low-copy number, I would not mind going for 24 hours. I have done 8 hours sometimes but I knew that my plasmid is high copy number and I got enough DNA.

If you don't know give a try and see what results you get. RUn 2 cultures side by side.


if its high copy and the culture is turbid, you can get DNA after only 6 hours, it just will be a lower amount....I have done this in the past when trying to get things done FAST....warren..



#44863 3 way ligation help needed

Posted by Warren on 14 November 2009 - 06:30 PM in Molecular Cloning

dephosphorylate your second fragment (the one with the xhoI and notI sites)...

warren..



#42186 Viral genome sequencing

Posted by Warren on 02 November 2009 - 04:11 PM in Molecular Biology

I am working to sequence negative strand RNA viral genomes de novo (i.e. not the specific-primer based approach that works so very well) and am having trouble with host contamination. In fact I began the project expecting data with ~90% host sequence, but now have ~99.5% host and less than 0.5% target sequence! Since at this rate a full lane of Illumina sequencing is not enough to provide a good draft sequence for a 20Kb genome!

Current protocol (in brief):
1. Virus is grown up in Vero E6 cells and the Trizol cell lysate shipped here
2. Chloroform extraction of the Trizol
3. cDNA synthesis from total RNA
4. Illumina/454 library prep (adaptors added, etc) and sequencing

I've thought about shearing up some monkey (the cell line is Vero E6) DNA, binding it to streptavidin beads. Using those beads to pull out the host DNA and only synthesizing cDNA from the unbound nucleic acid. Unfortunately Ive never made beads like this (although the idea sounds simple)any protocol suggestions for bead prep or other avenues to reduce the amount of host sequence in the final output?


Thanks in advance to any responses!


If you plan on using beads I would definitely not use them the way you are suggesting. Presumably, your samples are RNA (if not there are certainly ways of getting rid of DNA) -- if you tried to shear up host DNA you have big problems including: less than 2% of it will be "coding" sequence, of that only 50% will be able to bind RNA (the complementary sequence) and then surely not all the genes are expressed. So only a tiny percentage of your beads would correspond to any RNA in the sample. Then you have to take into account that there could be LOTS of certain RNAs yet your only getting a couple sequences per cell of DNA (ie, you can makes lots of mRNA from one gene!) so you would need tremendous amounts of sequences bound to beads to even make a slight dent in the population of mRNAs in your sample. If you are stuck on using this, your pretty much have to start with cDNA to even have a chance at working.

However, I think that kind of technology would be better to use to pull out your sequence of interest, rather than remove the huge amount of host nucleic acids. If you have regions of the virus genome that are well conserved than you could design beads to bind them, wash out all the other crap then elute your viral RNAs. I don't think those beads need a very large sequence to bind.

Another approach would be to take advantage of the giant size of your RNA of interest. You could try protocols to specifically precipitate high molecular weight RNA, which you could tweak, or even some kind of column -- since your RNAs are so huge compared to almost anything else in there, you would not have to be very precise to get it to work. You may even be able to find some spin columns that will not allow your huge RNA through so you can wash out almost everything else.

warren..



#40784 Help! Double restriction digestion failed....

Posted by Warren on 22 October 2009 - 04:24 PM in Molecular Cloning

Hello Fellas...

I start my double restriciton digestion few weeks ago and now I am totally stucked.

I have cloned an insert with into a TA vector pcr2.1. I have sequenced it and confirmed that my insert is correct. My insert carry a NdeI RE site.

then i try to digest my vector with NdeI and HindIII (which is on the TA vector). I used double digestion system and i have tried several approach to optimize my plasmid amount:

1) a 50 ul reaction system:
1ug of plasmid
1ul of NdeI enzyme
1ul of hindIII enzyme
5ul buffer 2 (NEB)
up to 50ul with water

2) a 50 ul reaction system:
2ug of plasmid
1ul of NdeI enzyme
1ul of hindIII enzyme
5ul buffer 2
up to 50ul with water

3) a 50 ul reaction system:
0.5ug of plasmid
1ul of NdeI enzyme
1ul of hindIII enzyme
5ul buffer 2
up to 50ul with water

I try to digest them overnight and 5 hrs, but all these gave me a very weak band or even no band after the digestion. I suspect it was due to incomplete digestion as the large band i oberve was pretty thick.

I have also tried sequential digestion as some said that the buffer may not be suitable for both enzyme. I used PCR purification to collect the first digestion during the sequential digestion then performed the second one. However, the gel result show a very weak band as the large cut plasmid and i couldnt observe my target band as it was like 300bp long.

My collegue told me to add 15ul of plasmid into a 50ul system, which is around 5ug of plasmid. I still working on that.

can anyone advice me what should i do? is that my protocol of digestion has problems? i prepare my plasmid by miniprep and yield around 300ng/ul plasmid after that.


Please advice! I am totally stucked in here right now. Thanks in advance!


Just a guess here, but because the conditions you described should digest the DNA no problem, I am wondering if you are sure the insert is in the CORRECT orientation to use the HindIII site on the pCR2.1 vector. With this vector and T/A cloning, the insert could be in either direction.....which means that if your NdeI site is on the side of the vector where HindIII occurs on pCR2.1, a digest will appear to only cut once even though both enzymes are working. When using the restriction sites available on the vector to recleave your clone, you have to make sure you are in the correct orientation, which usually requires screening several positive clones (sometimes more if you are unlucky :D to get one in the orientation you want. If you have other positive clones, test some DNA with the same enzymes from them....

Warren..



#40714 Restriction digest

Posted by Warren on 22 October 2009 - 08:11 AM in Molecular Cloning

Hi, I am new to do research. I found that normally the recognition sequence of restriction enzyme is from 5' to 3', for example, the recognition site for BamHI is

5' GGATCC
3' CCTAGG

If the sequence of my gene is

5' CCTAGG
3' GGATCC

Can I use BamHI to cut my DNA?


No.

Warren..



#40651 Best method for Mutagenesis?

Posted by Warren on 21 October 2009 - 04:56 PM in Molecular Cloning

Hi there,
I was wondering if anyone can offer any advice on the best method for mutagenesis experiments, which I am yet to embark on. I am looking at substituting 3amino acids with alanines at one site and 4 amino acids (substituting again with alanines) at another site, simultaenously. The gene in question is a 10.9kb gene encoding a cellwall anchored protein (with LPXTG motif) from Streptoccus gordonii. Is this gene too big for accurate and successful mutagenesis? If so, I could just mutate the 4kb N-terminus (TheC-terminus is 6.9kb and made up of 14 imperfect repeat blocks.) I have had alook through the posts and quite a few people are having problems with the kits (stratgene etc.) Is there another way other then bought kits? Also are there any 'hot tips' for mutating my monster gene.

Thanks!

Helen


Yeah you can just use PCR. I would just make subclones containing smaller fragments and do the mutagenesis on them, then subclone back into the full-length. It might sound like more work, but its really not if you can find convenient restriction sites, plus you can sequence your whole PCR-generated sequence from the subclone (alot easier than sequencing an entire 11 kb region to assure no unwanted mutations!) Warren..



#40650 ligation for 24 hrs in 4*C?

Posted by Warren on 21 October 2009 - 04:50 PM in Molecular Cloning

Hiya folks,
I have a question - can I leave my ligation reaction (pgemt) in 4*C for ca 24 hrs? I know they recommend an overnight reaction, but can anything go wrong if I leave it for 24 hrs?
best regards
kliper


from experience I am sure you can leave it at least a few days at 4 degrees, I wouldn't suggest it for long term storage though.....I have seen claims that too long a ligation time can cause problems in a buffer with PEG (something about creating giant oligimers and subsequent interference with transformation) but I haven't run into that problem. 24 hours should be about the same as 16 hours ("overnight") at 4 degrees.

Warren..



#40647 Sequence management software (for Mac)

Posted by Warren on 21 October 2009 - 04:45 PM in Molecular Cloning

Does anyone know of a software to manage sequences/restriction sites/primer design for Mac that is affordable (for a phd student) and similar to MacVector?

I'm doing my thesis in a chemistry institute and there isn't really anyone to ask about molecular genetics (i'm the only one doing this stuff..) plus if there's anyone they will be using PCs almost certainly.

Back when I worked for a pharmaceutical company (5 years ago) we used MacVector, so that's the software I know how to use.
Any input/suggestions would be greatly appreciated. Often I feel like I have no one to ask because of this research environment i'm based in. :P


Thanks a lot!!


Its been a while since I used MacVector (thats the one they didn't update for OS X right?) so I don't remember everything it can do, but a couple of programs that are FREE and I use alot are Serial Cloner and especially EnzymeX ..... you should be able to find these no problem and they are both pretty nice.

Warren..



#39767 problem with subcloning, please help!

Posted by Warren on 13 October 2009 - 03:19 PM in Molecular Cloning

Hai, thanks for your reply, we have a linker sequence inside the pBR322 that has these restriction sites, in fact i have cloned into this site ans have done several mutations before at this same sites (Not I and pac I), I am trying to make my final mutant and that is what is giving me the problem.


You have a "linker" or do you have pBR322 with a 2 kb insert that you are using? I can not see how you can cut a 2 kb chunk out of pBR322 without removing either the origin or replication or the tetracycline resistance gene (or part of it). Since you said you are using tetracycline to screen, I assume you are not cutting the gene out? Anyway, one thing I can say is that if you look at your ligation, and you see alot of a band that is double your PCR product size, but not bigger bands (ie, 3, 4 etc of your insert) it suggests only one of the enzymes is cutting your PCR product. This could explain your results.

Warren..


PS, there is an easy way to test this, perform a ligation with your digested PCR product only and run it on a gel....



#39766 problem with subcloning, please help!

Posted by Warren on 13 October 2009 - 03:16 PM in Molecular Cloning

Hai, thanks for your reply, we have a linker sequence inside the pBR322 that has these restriction sites, in fact i have cloned into this site ans have done several mutations before at this same sites (Not I and pac I), I am trying to make my final mutant and that is what is giving me the problem.


You have a "linker" or do you have pBR322 with a 2 kb insert that you are using? I can not see how you can cut a 2 kb chunk out of pBR322 without removing either the origin or replication or the tetracycline resistance gene (or part of it). Since you said you are using tetracycline to screen, I assume you are not cutting the gene out? Anyway, one thing I can say is that if you look at your ligation, and you see alot of a band that is double your PCR product size, but not bigger bands (ie, 3, 4 etc of your insert) it suggests only one of the enzymes is cutting your PCR product. This could explain your results.

Warren..



#39688 Lowered extension temps with Phusion

Posted by Warren on 12 October 2009 - 07:48 PM in PCR, RT-PCR and Real-Time PCR

Normal extension temperatures do not work for very high AT regions. I discovered this the hard way, but then discovered that this was a known effect. See PMID 8628694,
Su96, Reduced extension temperatures required for PCR amplification of extremely A+T-rich DNA. I would try Betaine instead of DMSO.


Thanks, I had actually gotten the idea from a few of your old posts, and had subsequently read that paper....I am surprised that this isn't included in literature for polymerases! I am curious though, why would you try betaine rather than DMSO? I'll have to order some betaine, the DMSO I already had. BTW, for whatever reason, it seems like I could get iTaq to polymerize through this AT rich region but not Phusion, which is also weird. Warren..


Just as a follow-up, in case anyone reads this thread for advice in the future: the temperature I ended up using with the Phusion Hot-Start is 64.5 (in this case I am just doing two cycle amplification, 98 and 64.5). It seems as though 1 min per kb in this case is not quite enough, for a 1.5 kb fragment 2 min worked well. Of course there is no separate annealing step so the 2 min includes annealing as well. Anyway, Phusion will actually amplify these AT rich regions extremely well at 64.5, just gotta give it enough time to do the extension. Warren..



#39687 Doing mutagenesi for large plasmid or articificial chromosome

Posted by Warren on 12 October 2009 - 07:15 PM in Molecular Cloning

I would subclone a smaller fragment into a regular vector, do the mutagenesis, then subclone it back in.

Warren..



#39684 problem with subcloning, please help!

Posted by Warren on 12 October 2009 - 07:03 PM in Molecular Cloning

Hai all,

I have been having this cloning problem for a while now...almost three months!

I am trying to replace a fragment of 2 kb from pBR322 vector, have done the same procedure several times before to get various mutants and has worked out so well before .... this will be the last one and it is this one which is causing trouble. here are the details:
the fragment to be replaced is 2 kb in size, has PacI and NotI as the restriction sites on either ends. This is what I do: I digest the plasmid (pBR322) using pacI and NotI (4 hrs digestion, 1ul of each enzyme, 4ug of DNA in 100ul reaction) this releases a 2kb fragment which i clean it out using gel cleaning technique (Qiagen gel purification kit). I have a 2kb fragment with PacI and Not I sites at its ends as a PCR product, also I have cloned the same in a Topo pCR4.0 plasmid. I digest this fragment and purify it. Later I ligate the digested pBR322 with this fragment. Ligation is done overnight at 16C using t4DNA ligase from NEB. I do the transformation in DH10B cells. I am always betrayed...there are no colonies in the plate the next day...it happens all the time....to make sure that the DH10B cells are okay, I did a control transformation...it worked...so the DH10B cells are fine...there is no problem with ligase as people in the lab using it get results!.....I did see something weird in my ligation ...the other day before transformation, I ran a little bit of my ligated product in gel....it was weird...i saw the digested plasmid, my insert and another fragment which was twice the size of my insert...is it possible that the insert got self ligated? or is there something else going on? I am completely frustrated...please help me!

thanks in advance


Are you sure of your primers ? I had the same problem with bad primers. Also, dou you have sufficient space around your resctriction sites on your insert ? Some enzymes need some extra bases to "sit" and cut...

You are talking about TOPO, did you try to get your insert out of it and load it onto a gel ?



Thank you for your msg, I had used the same primers before to make other mutations and has worked well, also I did try to digest the insert from TOPO and the size was fine. I forgot to mention about one more thing....I had gotten couple of white colonies sometimes, they are literally white colored and grow in tetracycline, show the size similar to my plasmid DNA but nothing comes out on sequencing. I am kind of frustrated at this stage.


pBR322 does not have either a PacI site or a NotI site. Try running an undigested sample of DNA next to your "digested" sample and they should look the same, unless you are getting some star activity. What your trying to do won't work.

Warren..



#37879 Gene insertion without desired unique restriction sites

Posted by Warren on 27 September 2009 - 12:43 PM in Molecular Biology

Hello friends,

while working on a cloning experiment i came across a problem.
I have to clone a gene into a binary vector that has been modified and contains only two restriction sites-NcoI and XbaI.
I need to use only this one to clone some genes that have either of the two or both the sites internally.
How can i use the same vector,ligate my insert into it and prevent my gene from being chopped off on restriction digestion with the above enzymes?


Break out your New England Biolabs catalog and look up compatible cohesive ends. For example, I know SpeI and NheI (both common enzymes most people have) are compatible with XbaI -- so if your gene doesn't contain these sites, then those could be used. The compatible ends for NcoI are less common enzymes, but then again, you could use any enzyme and blunt this end, and blunt the NcoI site; with a single cohesive end, the cloning is still pretty easy. Warren..




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