Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!

xyz's Content

There have been 23 items by xyz (Search limited from 30-September 19)


Sort by                Order  

#29282 cloning problem

Posted by xyz on 15 July 2009 - 09:05 AM in Molecular Cloning

Hi everyone,
I am having problem in cloning of PCR product (1.4 kb) into vector (PUC19-2.6 kb). I am using BamH1 restriction sites for cloning. After ligation and transformation I am not getting any colony in both ligation product and negative control as well. I tried various combination of insert: vector, but futile. i didn't get even a single colony. Any suggestions? I am working on this from a month------------------



#26854 PCR screening of transformed bacterial colony

Posted by xyz on 16 June 2009 - 10:00 AM in Molecular Cloning

Thanks for reply

I usually screen by doing a colony PCR with one primer that starts from the 5' end of my insert and a reverse primer that is from my plasmid. The volume would be 30ul. Usually load like 3 to 5ul of the PCR products on the gel. and, if you want an easier time, you can use GelStar, which is pretty sensitive and allows the band you want to shine up very easily under blue transilluminator light or UV light.




#26853 PCR screening of transformed bacterial colony

Posted by xyz on 16 June 2009 - 09:59 AM in Molecular Cloning

Thanks a lot.

Which primer you use depends on what you want to do with the plasmid. If you want to use it for normal cloning of your gene, you can use internal primer like yours. If you want to overexpress your gene, and you need the correct orientation, you might want to use a forward primer on the plasmid and a reverse primer on your gene. Positive colonies will have the right orientation.
20ul reaction mix is sufficient to screen colonies. You need to pick the colonies and put them into the H2O needed for your PCR reaction (15ul?), and then into LB medium until your PCR finishes. Then you can conveniently expand your positive colonies. I strongly recommend to use a positive control, e.g 5ng of a vector containing your gene.
I usually use 4ul of PCR product (30cycles) for the gel. If you have used 10ul DNA ladder and got faint staining, it seems your gel does not contain EtBr...
Cheers,
Minna




#26777 PCR screening of transformed bacterial colony

Posted by xyz on 15 June 2009 - 02:08 PM in Molecular Cloning

Hello everyone,
I had transformed the desired clone and got nice separated colony. Before sequencing them I want to screen the colonies. For that I chose the forward primer of my gene and reverse primer lies in the middle region of gene(Is this a good strategy?). Is 20microloiter PCR reaction mix is fine to screen the colonies? After PCr amplification how much volume should I load in the thin combed agarose gel? I tried with 10microliter of DNA ladder in the gel, but very faint resolution. Any suggestions--------------



#26615 No product after restriction digestion

Posted by xyz on 12 June 2009 - 09:12 AM in Molecular Cloning

Thanks

Can you please provide me the exact protocol of it.
Thanks

It sound good but does it cause any problem in firther cloning assay
thanks

Some of the groups in my institute are moving away from kits etc completely, and just crush the gel slice, make up to 1 .5 ml with TE and leave in the fridge overnight. The next morning they spin down the agarose, remove the S/N and do an ethanol precipitation. Slow, I know, but it's faster in the long run. If the DNA product is smallish, use ammonium acetate rather than sodium acetate: 1/3 vol 7.5M ammonium Ac, 2 volumes of the new volume EtOH.

There shouldn't be any problems at all. Once you have done the EtOH precipitation, it's as clean as DNA prepared by kits. Just make sure you get rid of trace EtOH, by leaving the tube open and inverted for a few minutes. You can also heat gently for a few minutes to make sure.

The protocol is pretty much as I wrote above. Make sure the gel is diced into small pieces, as this speeds up the diffusion process into the TE.
The agarose is spun down hard for 10 minutes and the S/N is simply decanted off. Precipitation can be done with either sodium or ammonium acetate. If your yields are expected to be low, co-precipitate with glycogen, using standard procedures.




#26530 No product after restriction digestion

Posted by xyz on 11 June 2009 - 09:02 AM in Molecular Cloning

Thanks a lot. what do you suggest for the vector DNA ratio.

Can I do like that !!!because I have to ligate it before and for that minimum amount of DNA is required 100 ng.

Although you don't see anything on the gel: Just try to clone it.

And if that doesn't work: Avoid using gels and kits! Make phenol-chloroform-extraction or only ethanol-precipitation instead...


100 ng is waht the protocol says, and that is probably the best way to make sure that ligation does work.
But that doesn't mean that it doesn't work below that.
I have often ligated fragments that I haven't seen on the gel, and most times it worked out well (maybe I had just 1 out of 24 instead of 12 out of 24 right clones).
Just take less amount of your vector to make sure your vector-fragment-ratio isn't too bad.




#26486 No product after restriction digestion

Posted by xyz on 10 June 2009 - 08:32 PM in Molecular Cloning

Can you please provide me the exact protocol of it.
Thanks

It sound good but does it cause any problem in firther cloning assay
thanks

Some of the groups in my institute are moving away from kits etc completely, and just crush the gel slice, make up to 1 .5 ml with TE and leave in the fridge overnight. The next morning they spin down the agarose, remove the S/N and do an ethanol precipitation. Slow, I know, but it's faster in the long run. If the DNA product is smallish, use ammonium acetate rather than sodium acetate: 1/3 vol 7.5M ammonium Ac, 2 volumes of the new volume EtOH.

There shouldn't be any problems at all. Once you have done the EtOH precipitation, it's as clean as DNA prepared by kits. Just make sure you get rid of trace EtOH, by leaving the tube open and inverted for a few minutes. You can also heat gently for a few minutes to make sure.




#26441 No product after restriction digestion

Posted by xyz on 10 June 2009 - 09:56 AM in Molecular Cloning

It sound good but does it cause any problem in firther cloning assay
thanks

Some of the groups in my institute are moving away from kits etc completely, and just crush the gel slice, make up to 1 .5 ml with TE and leave in the fridge overnight. The next morning they spin down the agarose, remove the S/N and do an ethanol precipitation. Slow, I know, but it's faster in the long run. If the DNA product is smallish, use ammonium acetate rather than sodium acetate: 1/3 vol 7.5M ammonium Ac, 2 volumes of the new volume EtOH.




#26440 No product after restriction digestion

Posted by xyz on 10 June 2009 - 09:53 AM in Molecular Cloning

Can I do like that !!!because I have to ligate it before and for that minimum amount of DNA is required 100 ng.

Although you don't see anything on the gel: Just try to clone it.

And if that doesn't work: Avoid using gels and kits! Make phenol-chloroform-extraction or only ethanol-precipitation instead...




#26334 No product after restriction digestion

Posted by xyz on 09 June 2009 - 10:03 AM in Molecular Cloning

Thanks.
Star activity depend on units of enzyme to conc of DNA , glycerol concentration and some other factors as well.I am using only 1 microliter of enzyme and that gives only 20units/microgram of DNA, so I am assuming that star activity is not giving problem to mu experiment.
If is there anything that I am not getting about star activity

No I didnt do that. Just after purification digested the product

did u run a gel to check if you have your products after purification and before the restriction digestion?

you should have done that because it might save much time for you.
ps: bamhi has star activity from time to time at a high concentration.




#26330 No product after restriction digestion

Posted by xyz on 09 June 2009 - 08:53 AM in Molecular Cloning

No I didnt do that. Just after purification digested the product

did u run a gel to check if you have your products after purification and before the restriction digestion?




#26329 No product after restriction digestion

Posted by xyz on 09 June 2009 - 08:51 AM in Molecular Cloning

Hi
Thanks for suggestion. What is the rationale for native Page( poly acrylamide GE). and one more question is that checking the conc with spec (Nanodrop) makes any sense?

Just one suggestion from my side.

As after purification with Qiagen kit, the concentration lowers , so check the concentration, run the gel. If you see the band, Then take appropriate amount to digest.
After digestion, sometimes instead of running agarose gel, for small DNA fragments run Native Page. It helped me a lot.

Thanks,




#26254 Simply Beautiful!

Posted by xyz on 08 June 2009 - 12:41 PM in Chit Chat

AMAZING

It's got your eyes...:( (or is it ears?)





this one's really electrifying:

Posted Image




#26253 No product after restriction digestion

Posted by xyz on 08 June 2009 - 12:37 PM in Molecular Cloning

Hi all,
I amplified my PCR product and it gave a nice band (4 microgram conc around), after that I puified it with qiagen kit and digested it with BAMh1 enzyme , after that I didn't get any band on gel. What could be the reason. I tried both qiagen I and Qiaex II kit for purification but no yield. Need suggestions.



#26061 restriction digestion of PURIFIED PRODUCT

Posted by xyz on 05 June 2009 - 11:41 AM in Molecular Biology

Thanks
I had run the digestion and made the total voume 30[ul] , so i can directly run it on gel. I have one more question that how much amount of DNA is required for digestion? is it 1 microgram?
Thanks again

What's your confusion and what's ur approach?

Tell us ur reaction . I want you to come up with something. then we will look at the problem.

Vector digestion and PCR product digestion share the same reaction. nothing special. just use the standard reaction condition..

There's really no FIXED thing around here. Some ppl like to use more some swear to using less of the same compound.

BSA conc. is normally written on the manual so i don't know why u ask this.
BamHI just use 1ul for whatever .

You can't determine the concentration at all so what? just run the digestion as usual. 1U = 1ug. but 10 fold of that is still fine

Seriously just run the reaction . u will feel better.

regards,

Ming




#25892 restriction digestion of PURIFIED PRODUCT

Posted by xyz on 03 June 2009 - 12:23 PM in Molecular Biology

Because I am not able to decide the concentrations at all, so-------------. I read the manual but still I have some confusions and I am not sure about vector digestion?We use the biolab product for digestion. do u have experience for that?

Hi all,
I have purified my PCR product by QiaexII kit and by eye visualization I guees that the concentration of product is around 4[ug] in 40[ul] in total volume. Now I want to digest this PCR product and vector that already have any other gene in it and I want to replace it with my PCR product. I had BAMH1 restriction sites in PCR prodct and vector. So what to do for digestion? What concentration of enzyme,PCR product ,BSA should be used both for PCR product and vector. And how much final volume should I use?
Its really giving me a lot of headache to decide everything. Need help



It shoudln't be giving u headache.. seriously

just read the manual. follow the instruction . u be fine.
BamHi can't be heat inactivated efficiently.

I really don't know why this can give u headache.. . . .




#25869 restriction digestion of PURIFIED PRODUCT

Posted by xyz on 03 June 2009 - 07:52 AM in Molecular Biology

Hi all,
I have purified my PCR product by QiaexII kit and by eye visualization I guees that the concentration of product is around 4[ug] in 40[ul] in total volume. Now I want to digest this PCR product and vector that already have any other gene in it and I want to replace it with my PCR product. I had BAMH1 restriction sites in PCR prodct and vector. So what to do for digestion? What concentration of enzyme,PCR product ,BSA should be used both for PCR product and vector. And how much final volume should I use?
Its really giving me a lot of headache to decide everything. Need help



#25868 volume of DNA required (PCr product ) in agarose gel

Posted by xyz on 03 June 2009 - 07:42 AM in Molecular Biology

Thanks for information

I generally just go ahead and run 10 ul per well (5 if I'm really trying not to use up my pcr product) for a 12-well comb in a mini gel. The promega ladder that we use calls for 5 ul. I add about 3 ul of loading dye. Just go ahead and try it - you can always make more pcr product if you need to.

Hi,
Thanks
Means for a bigger well 30-35[ul] is sufficient? and how would i know the concentration of my product.[/u]

volume depends on the concentration of your products. The higher concentration, the less you need to add. Also, the bigger your well in width, the more you you need to add. For a PCR product that you are trying to clean up, I would load as much as you can get.




#25867 Problem with DNA extraction from 1% agarose Gel

Posted by xyz on 03 June 2009 - 07:39 AM in Molecular Biology

There might be some contamination in buffers!!!

I get horrible yields with the Qiagen kits. However. . . what are you trying to do with your DNA afterwards? I have had no problems taking the negative yields of DNA gotten from the Qiagen kits and ligating it into vectors and transforming with it. Plenty of colonies.




#25785 Problem with DNA extraction from 1% agarose Gel

Posted by xyz on 02 June 2009 - 10:00 AM in Molecular Biology

Hi,
I have tried with qiagen Qiaquick kit and it didn,t give me good yield (how dou check the yield?) So I was being suggested to use Qiaes2 kit. So i will do with that. so 1 one question I have is that in both of kits elution buffer are same or different? according to me its same 10 mMTris Cl PH 8.0, but as u said that both use different buffer?

I'm a little lost here and hoping that someone may be able to shed some light on a problem I'm having.

I'm trying to purify and isolate a PCR fragment (~1.8kb) by running about 10 - 20ul of my PCR product on a 1% agarose gel 110V ~15mins. So far I've tried three methods, Qiaex 2 gel extraction kit, Qiagen Qiaquick Gel Extraction kit, and using DAE81 filter paper to purify my DNA. In both Qiagen kits I get no yield after following the protocol exactly. The only variables are the length of time I dry the pellet in the Qiaex 2 kit and the elution buffer ie ddH20 pH 8 or Tris-HCL pH 8.

I've had previous issues with the elution buffer and have checked that twice now. I have a very strong PCR product band in the ug yield and even have tried diluting the PCR product in case the [DNA] was too high and somehow caused problems with binding. Washed and cleaned and rechecked my TAE buffer, gel, machine and reagents.

The DAE filter paper method gives me aprox 30% yield but as I'm attempting to do cloning after restriction digest and purification again my [DNA] is too low for ligation.

I'm attempting to use a new kit from another lab at the moment but if anyone else has had similar problems or any insight I would very much appreciate the help!! Thanks,




#25779 volume of DNA required (PCr product ) in agarose gel

Posted by xyz on 02 June 2009 - 08:21 AM in Molecular Biology

Hi,
Thanks
Means for a bigger well 30-35[ul] is sufficient? and how would i know the concentration of my product.[/u]

volume depends on the concentration of your products. The higher concentration, the less you need to add. Also, the bigger your well in width, the more you you need to add. For a PCR product that you are trying to clean up, I would load as much as you can get.




#25778 volume of DNA required (PCr product ) in agarose gel

Posted by xyz on 02 June 2009 - 08:19 AM in Molecular Biology

Hi,
Thanks for information, but I am naive in molecular world, so want to know that how would I estimate the concentration of my PCR product(DNA)

It hard to say, it depend or how much amplification you have. If you have a very good amplification, you can run 5-10 ul of your PCR reaction, better if you load everything. Use (again depend of the ladder you are using) for example 5 ul of 0.1 ug/ul ladder.
And very important it depend the kit you are using to purify your DNA, take account of the specification in the kit. For example, for QIAaquik kit: use up to 400 mg of agarose fragment, if you use more, you’ll recover very few DNA. Load up to 10 ug DNA/column, if you have more DNA than that, use two columns.
In few words, READ THE KIT'S MANUAL BEFORE DO IT.




#25725 volume of DNA required (PCr product ) in agarose gel

Posted by xyz on 01 June 2009 - 02:36 PM in Molecular Biology

Hi,
I am doing the cloning experiment. SO i have amplified my product thru PCR and after that visualized it thru 1% agarose gel. Now i want to run on gel ( with large combo size ) so that I can purify it. I want to know the volume of PCr product and DNA ladder required to load in a gel




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.