Molecular Cloning Method Discussion

overlapping PCR protocol

Thu, 09 Feb 2012 14:08:25 +0000

Hi all!

I want to make an overlapping PCR with 2 fragments, one has 2200bp and the other has 6000bp. The two fragment share 45 pb homology to their end.

I know that, theorically, I can generate a ''new'' fragment of 8200 bp, but I have no idea of the experimental condition that I should use to get succes! If someone can suggest me some experimental conditions, such as amont of DNA to put into my PCR, PCR amplification conditions and polymerase to use, it would be very helpfull.

thank!

cloning an AT rich gene

Mon, 06 Feb 2012 16:17:46 +0000

Hi,
I am new to cloning and facing a big problem.
I am trying to clone an AT (80%) rich gene from Plasmodium.
I have PCR amplified the target gene, extracted the band from the gel, purified it with Qiagen Gel extraction kit.

I run it on a gel to estimate the concentration and ligated it into PGem plasmid using the PGem Teasy kit in a 1:1 molar ratio (insert to vector).

Transformation looks o.k.
I did colony PCR with insert primers and got a strange size band, 1.2kb instead of 550bp? what could this be?

the other problem is when I try to sequence it with SP6 and T7 primers I only get the vector sequence.

did the ligation not work? how could I test it?

thanks

Cloning two genes in the same plasmid

Mon, 06 Feb 2012 14:54:37 +0000

Hi all,

I would like to try co-expression of two genes in one plasmid under different promoters.
I'm looking to pFast bac Dual plasmid then I've seen two promoter p10 and pPH with two MCS, and they are at opposite direction.
I don't know if the expression will be into two directions. I mean how to choose the restriction enzymes (which one, forward and reverse with the other promoter) they seem to me in opposition.
Could you please help.

Thanks

How to wipe out fragment of dead cells from the suspension cells?

Mon, 06 Feb 2012 06:01:26 +0000

As show in the title, Many dead cell fragments were in the suspension cells, I can't wipe out, who have good method to work out this question?

why my solution II turns cloudy during plasmid extraction?

Sat, 04 Feb 2012 18:11:12 +0000

I use conventional Sambrook's method for plasmid extraction, but recently when I make solution II with SDS and NaOH the solution turns cloudy. I also noticed the my DNA pellet is getting smaller. has anybody ever have the same issue ?

Problem: Cloning DNA shuffled band

Fri, 03 Feb 2012 14:37:02 +0000

Hi!
I am currently doing DNA shuffling. The shuffling procedure itself is not a problem for me. I have optimised the initial PCR, DNAseI digestion, PCR without primers and reamplification with primers. Up to this point, everything seems to be going great. Afterwards I digest the shuffled DNA in he usual way and I try to clone it into my vector.
And here comes the problem: I am never able to get good colonies (tested by restriction digest).
My controls indicate that the restriction enzymes, ligase, etc, are working well. The electrocompetent cells are good, since I get plenty of colonies, but when I get to analyse them, they are all incorrect. Not a single good colony.

I have cloned a lot during my thesis, so I think I have enough experience. This one should be very easy. However, whenever I try to clone shuffled fragments, I have this problem.

Does anybody know wheher there is something special or something different to a normal cloning, that I should do in order to clone the shuffled fragment?

Thanks in advance!

shRNA to primary keratinocytes

Thu, 02 Feb 2012 08:47:27 +0000

Hi!

I'm wondering how I can deliver shRNA into primary keratinocytes. I cannot use a growth medium containing lentivirus, because it contains serum and my cells differentiate when exposed to it. I've read about many kits that come ready (plasmid+transfecting agent, plasmid+virus), but the problem is that we have already ordered the plasmid kind of withoud wandering what to do next.

Does any chemical reagent that easily transfect the cells and doesn't kill them come to your mind? They have to be in a good condition because afterward the cells will be subjectet to quite an unpleasant treatment.

What do you think about applying lentivirus-containing medium onto the cells, centrufuging the plate and changing medium to keratinocyte growth medium after 1 hr (maybe the cells won't "notice" there is something wrong with the medium)
http://jcs.biologists.org/content/120/2/330.full <- they did this with retrovirus

Enzymetic digestion with ApaI and HincII

Thu, 02 Feb 2012 08:45:01 +0000

Dears, I amplied a new tag with primers having recognition sites for ApaI and HinCII, gel extracted the amplified fragments and then did the digestion with ApaI (incubation temperature at 25C) for three hours and kept it at 4C without heat inactivation. The next day I digested the same sample with HincII (ApaI and HincII both can work 100 % in NEB Buffer 4) at 37C for three hours and then heat inactivated. My question is since I didnt heat inactivate the sample after digestion with ApaI, was ApaI still active at 4C?.......basically I need to cut the extra fragments amplied with restriction enzymes and then to insert it into my vector.....

Ligation gel picture interpretation

Wed, 01 Feb 2012 17:05:36 +0000

Hello, I am trying to insert a 2 kb PCR fragment with EcoRI/XbaI sites into a pET32a vector (5.5 kb) that is digested with EcoRI/XbaI. I did a 3 hour ligation.

Control: Vector + ligase - - - - This gave approximately 50 colonies
1:3 Vector:Insert - - - - - - again around 50 colonies
1:8 Vector:Insert - - - - - around 60 colonies

I am worried about all of the self ligation that occurred and I doubt that any of my colonies on experimental plates are the clone I am looking for.

I ran some of the leftover ligation on a gel and would like some input on the multiple bands I observe in the vector + insert ligation reactions. I will be doing minipreps and XbaI/EcoRI digestions tomorrow so I will find out then if it worked for sure, but is the expected 7.5 kb vector+insert band in the smear?

lane 1: marker (12000, 8000, 6000, 5000, 4000...)
lane 2: control (vector + ligase)
lane 3: 1:3 vector:insert
lane 4: 1:8 vector:insert

Also, if you have any suggestions to limit self-ligation since my control plate had as many colonies as the actual ligation they would be appreciated. I have not used SAP or the likes for the vector since I have incompatible ends.

Thanks in advance

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strange subcloning problem

Wed, 01 Feb 2012 08:38:41 +0000

Hi

I have a strange problem in my ligation

We ordered a synthetic gene from Genscript which is supplied in puc57 vector. I subclone the gene from puc57 into our self-constructed expression vector using NgoMIV and BsRGI sites. Below are our procedure

1. digest the insert and vector with BsrGI and then NgoMIV
2. gel purify the insert (~800bp) and vector (~10kb)
3. ligation in 1:3 or 1:5 ratio at 4C overnight or 16C for 4 hours
4. purify the DNA and then transformed in DH10b cells purchased from invitrogen

I usually get ~50 colonies in the ligation while my control without insert has ~0 to 10 colonies only.
however when i miniprep and check my plasmid, most of them are background from my insert prep, which is the puc57 vector with my synthetic gene in it.

i repeated it for many times but still no positive clones, I want some suggestion of it!

Cloned sequence is not predicted size by western blot

Tue, 31 Jan 2012 23:03:41 +0000

Hi, we sub cloned a DNA sequence into our vector of choice. The sequence subcloned was predicted to give a protein molecular weight of 36 kda. When western blot was performed of transduced cells ( expressing the gene of interest), we got a very good western blot signal. However the molecular weight of the protein detected was 48 kda (using a nuMber of different antibodies). Does anyone have an idea of why our cloning is generating a protein that is 10kda heavier than that is predicted by its DNA sequence. It seems unlikely to be phosphorylation etc.. We believe we have completely denatured the protein lunate, including further 'linearisation' using urea.
Cheers
Alex

Trouble cloning E.coli cells with pUC18 and insert

Tue, 31 Jan 2012 17:51:11 +0000

Hi all,

I hope someone can help. I am currently having trouble cloning 7 PCR products (between 1.5kb and 2.5kb) into E. coli MKH13 using the pUC18 plasmid (2686bp). I use positive and negative controls during transformaion (i.e electroporate just the plasmid (no insert) and electroporate just the cells without plasmid). The PCR products show good strong bands on a gel after purification so I know this is not the problem however there are many other steps to consider when trying to find the source of the error as outlined below.

I was getting no growth on any plates when using LB with 100ug/ml Ampicillin. I have dropped the Ampicillin concentration to 50ug/ml as the literature suggests that this concentration of Ampicillin should suffice. However now I am getting growth on LB with 50ug/ml but when I tried extracting the plasmid I found there was no plasmid on the gel suggesting that perhaps the wild type was growing at 50ug/ml. I did an MIC on E.coli MKH13 also and got an MIC value of 1ug/ml suggesting that 50ug/ml would be more than enough to inihibit the growth of this organism without the plasmid and insert.

Also I am making the cells electrocompetent myself (using the glycerol wash method) as I don't think you can buy competent MKH13. I am strictly following this method and ensuring that the cells remain cold at each stage however i'm wondering if the electroporation transficiency is playing a role. I have plated the electrocompetent cells on LB and got a high colony count (TNTC at 10-5) however although there is a large number of cells it does not necessarily say that the cells are electrocompetent so I will need to electroporate the cells with plasmid and do a count again. Does anyone know if you can buy electrocompetent MKH13 if this doesn't work?

Apologies for the essay! Hope someone can shed some light on this! I read on this forum today that greater than 4ul of ligation mix can actually inhibit transformation. I was using 5ul of ligation mix and 50ul of competent cells so I will try it with 2ul ligation mix next. Any other suggestions/comments would be much appreciated

Problems with pmiR-report vector

Tue, 31 Jan 2012 07:45:04 +0000

Hello:

i've been trying to use pmiR-report vector to clone a 2.7kb i3'utr and my problem is that even if I'm getting cells with the clone, all of them have any problem. The first time I got the colonies I find out that in one i had lost the luciferase gene, the second time it was the CMV to be lost and two days ago i found a plasmid which had like another extra 3kb in their sequence.

I've worked with other vectors and it seems to me that the error ratio here is much more higher that as usual!

Does anyone have an idea about what's going on? i thought that maybe it would be a problem with the compatibility of the e.coli strains i was using, maybe something about recombination. I've used JM109 from promega the first time, and now i was using DH10B. I haven't found any restrictions about the e.coli strains in the manual so right now i'm pretty desperate.

Thanks

Ligation of insert to itself problem

Mon, 30 Jan 2012 19:14:03 +0000

Im having issues ligating my insert into pet-28a. I get fine digestion using NdeI and BamHI but when i go to ligate it seems as though my insert is being ligated to itself due to the appearance of multiple bands in my gel, seemingly being multiples of the insert. What should i do to get it to ligate to my vector instead of itself?

double digest problem

Sat, 28 Jan 2012 11:02:00 +0000

Hi
i did a double digest with Ecor1 & BamH1 with a 7Kb plasmid sample prepared through miniprep but not purified.
in 10ul reaction volume i used 2 ul DNA ,1ul10xBSA ,1ulNEbuffer4 and 0.2ul of each enz.
got no expected fragment . these were plasmids taken from transformants of the ligation mix of backbone + fragment.
pls help

Difficulty in expressing fusion protein

Fri, 27 Jan 2012 23:24:12 +0000

Hi,
I am trying to make a fusion protein. My model organism is zebrafish. Initially I tried to express my protein with Mcherry at its N terminal. I could see the expression of Mcherry in my wild type fusion protein. However, when I made a mutant fusion protein, I couldnot see any expression of Mcherry. Then I repeated the same procedure with GFP as a fusion and got the same result. I tried to check if the protein is expressing by western, but the antibody doesn't work. I tried using a GFP antibody, and got a thin band with my Wild type fusion protein but the size of the band was same as that of GFP alone.

Can anybody help out?

Thanks

ligation - DNA present when run on gel, absent when using PCR!

Thu, 26 Jan 2012 21:53:20 +0000

I am trying to insert 4kb into pGEM T-easy vector. I used Phusion polymerase, PCR purified, and A-tailed product. Went on with ligation. Ran ligation mixture on gel to visualize DNA and expected size bands were present. Then ran PCR on the ligation mixture using M13 primers (suggested to me by PI) and I have no bands. Transformed and have yet to find a positive clone using PCR. Screening with M13 primers. Should I use my sequence specific primers? Or use in in the vector and in the insert? I don't want to do this as I will have to set two reactions per sample to test directionality.

Then I tried inserting a different gene that was 2kb. I performed the same procedures and was able to see my DNA by running the ligation mixture on the gel AND by PCR. Then transformed. Used colony PCR to test six colonies and five out of six had the insert. So should I give up searching for my experimental insert since it did not amplify using PCR? And is colony pcr the best way to screen many colonies quickly?

Any advice is appreciated.

Plasmid recovery problem

Thu, 26 Jan 2012 21:19:28 +0000

Hi,

I'm having major issues with plasmid minipreps. I've grown my culture from glycerol stocks and the overnight culture gives me a very dense culture (5 mL). I've spun this down and did my miniprep using the Invitrogen kit and I'm getting very very low concentrations (~35 - 70 ng/ul). I thought maybe the kit was old, so I got a new and I'm getting the same results. I have no idea why this is happening.

Help!

Restriction Digest Problem

Thu, 26 Jan 2012 19:11:23 +0000

I have been trying to digest my pET32a vector with both EcoRI-HF and XbaI. The restriction sites are approximately 550 bp apart from one another. I have tried 2 hr, 4hr, and overnight digestions. Below is my reaction mixture.

pET32a vector (3 or 1 µg) 28 µL
NEB Buffer 4 5 µL
BSA (100 µg/ml) 0.5 µL
EcoRI-HF (40 units) 2.0 µL
XbaI (40 units) 2.0 µL
H₂O 12.5 µL

TOTAL 50 µL

Attached is the gel image. Lanes 2 and 3 are EcoRI only for a linear control. Lanes 4 and 5 are 3 ug vector double digest, and lanes 6 and 7 are 1 ug vector double digest. All digests were overnight. You can see that the correct size fragment is present around 600 bp (200 to 12000 bp ladder) but it is very incomplete

Does anybody have an idea as to why I have incomplete digestion? I think it might be the plasmid prep. The uncut plasmid hardly migrates on a gel as shown in the Picture 1 figure in lane 2. The plasmid could be nicked, and that is why it wont migrate but it still should be digested.

It seems as if no matter how much enzyme I add or how long I digest, I do not get any significant digestion.

Any advise? Tomorrow I will be doing a new plasmid prep.

Thanks

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Recombination protocol needed

Wed, 25 Jan 2012 15:43:27 +0000

Hi all,

I want to clone 2,2kb fragments into 15 kb vector by bacteria recombination, because I have tried several time by ''classic'' clonage (restriction enzyme and ligation) and it doesn't work.

Thanks,