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Troubleshooting with restriction enzymes during cloning

cloning pgem t easy restriction enzyme subcloning

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#1 liligompac

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Posted 25 April 2018 - 03:31 PM

Hi!

Can someone help with my restriction digestion problem with pGEM T Easy vector?

I am trying to produce recombinant GH, so the first step was to amplify by PCR the gene, besides the sequence of the gene, the primers also contain restriction sites for EcoRI 5' and NotI 5'. After the PCR, the fragment was purified by gel extraction, then the fragment was ligated with pGEM T-easy, I grew up colonies with the product ligated, then I made a miniprep. But when I want to cut the pGEM-T easy with EcoRI and NotI, the restriction digest is not produced, I expect 2 bands on the gel, one of 650 pb (the insert) and the other of 3000 pb. At the same time I have another vector (pCAG with GFP, I want to use this plasmid to produce my desired protein) that I digested with the same enzymes so I can remove the insert of GFP (600 pb), but in this case the digestion worked. Another information to add is that the concentration of both plasmids for the  digestion was 3 micrograms and I added 2 microlitrers of each enzyme. I don't know if it has to do with the concentration of the plasmid or the enzymes. 


Edited by liligompac, 25 April 2018 - 09:15 PM.


#2 bob1

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Posted 25 April 2018 - 06:24 PM

There are a couple of things that could have gone wrong here.

 

Check that you placed the NotI site at the 5' end of the primer.

 

Try using less DNA and check the concentration of the enzyme - you typically need a 5-10 U for 1 ug of DNA. Diluting out the DNA can help to - see next point

 

Try a fresh miniprep or try re-precipitating the DNA - sometimes you get inhibitors coming through the extraction that can interfere with downstream steps. This is most likely when you didn't remove all the medium before lysis.



#3 liligompac

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Posted 25 April 2018 - 09:42 PM

I have the sequence of one colony (I chose it because it was the purest and the more concentrated of the six colonies that I chose randomly), it has the NotI site but it lacks the EcoRI, despite that, pGEM T-Easy contains both restriction sides at the places where I want to cut my insert, so there must be a cut. I wil try re-precipitating the DNA (but how should it be done?) and changing the concentrations of the DNA and the enzymes. Thanks for your suggestions.

There are a couple of things that could have gone wrong here.

 

Check that you placed the NotI site at the 5' end of the primer.

 

Try using less DNA and check the concentration of the enzyme - you typically need a 5-10 U for 1 ug of DNA. Diluting out the DNA can help to - see next point

 

Try a fresh miniprep or try re-precipitating the DNA - sometimes you get inhibitors coming through the extraction that can interfere with downstream steps. This is most likely when you didn't remove all the medium before lysis.



#4 bob1

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Posted 26 April 2018 - 06:18 AM

Re-precipitation is pretty easy - all you have to do is add some salt and ethanol or isopropanol. You would want the salt (NaCl works fine) to be about 0.5 mol/l and add a volume of 100% ethanol/isopropanol equal to that of the solution + salt mix. Let sit for 10 min, then spin down at max RCF in a microfuge. Remove supernatant carefully avoid touching the pellet, wash with 70% ethanol, re-spin down. Remove wash as completely as possible and allow pellet to air dry. Dissolve pellet in buffer of choice.



#5 liligompac

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Posted 26 April 2018 - 03:07 PM

I made the re-precipitation but the concentration of the dna was too low, also I didn't appreciate the pellet :(



#6 OldCloner

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Posted 19 February 2019 - 11:36 AM

Maybe you don’t have any inserts in the clones you picked (?)

 

First, you should be amplifying your PCR with a polymerase that adds A’s to the termini of the amplified fragments. Ordinary Taq polymerase will do so, but many of the “proofreading” polymerases do not add A’s; they create nice perfect blunt ends instead.  So check which polymerase you are using.  There are kits for “easy” cloning of blunt-ended PCR fragments if it turns out you are using a proofreader enzyme.

 

Second, if possible, avoid gel purifying your amplicon. Run just a small part of the PCR reaction on a gel to check its quality. If you are getting a nice single band product (optimize reaction if needed) you can best purify the fragment (from the remaining reaction) with one of the spin-column PCR clean up kits. That will get rid of left over primers/oligos/rxn ingredients from the PCR.  I used to amplify 25 ul PCR reactions, run 5 ul to check it, then purify the other 20 ul. You will likely have better luck trying to clone an amplicon purified this way. After purification, run a little bit of your purified product on a gel to see if it still looks good, before setting up your ligation.

 

You might even be able to get away with not purifying the amplicon at all. Check the instructions for your vector.

 

pGEM T easy vector appears to have a lacZ cassette for blue/white screening with X-gal in the medium. (And IPTG if needed, depending on the E. coli strain you are using).  Then you eliminate the empty vector background by NOT picking blue colonies (are you familiar with this kind of old fashioned screening?).  Is your vector really old? If so it might not work well.

 

Another suggestion: if you can change vectors is to try one of the Invitrogen (now Thermo) TA cloning kits.  They use a topoisomerase rather than a ligase, and they have a special system for “eliminating” (mostly) the background (recircularized empty vector)-containing colonies. They also make kits for cloning the blunt ended types of amplicons.

 

I hope these suggestions are helpful- good luck!


Edited by OldCloner, 19 February 2019 - 11:38 AM.






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