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Restriction Digest with pGEMT for TA cloning

cloning digestion Restriction enzymes PCR cloning pGEMT easy vector

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#16 Mashudu

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Posted 04 May 2018 - 05:00 AM

 I have a codon-optimized gene cloned into pUC57 with AvrII and XmnI overhangs. I want to subclone the gene into a yeast expression vector (pKOV410). The size of my gene is 1429 bp and the expression vector is 6352 bp. The overhangs are close to each other on the vector. I did a doble digest on the pUC57 to release my gene, ran a gel and extract my gene of interest using Promega gel extraction kit with a final elution volume of 50 ul. I get 20ng/ul concentration of my gene. I also did a double digest of the pKOV410, run and extract the linearised vector. The nanodrop concetration of the vector is 45ng/ul. I am using NEB ligation kit. I tried ligation at different environment (3hr RT, 16hr O/N, 4 degrees O/N). I take at least 3-5 ul of my ligation into 50ul JM109 competent cells which we just bought. I do a control with uncut vector and i get hundreds to thousands of colonies. The ligation kit is new and i made aliquots of the buffer the day i received the kit. The enzymes are from NEB & they recommend digestion for 5-15min, but it seams not to be enough, so i went for 1-3 hours, that works well. But i still dont get a single colony. One of the overhangs is blunt & the other is sticky. I have not tried sequential digest yet. The antibiotic resistance for the expression vector is Kanamycin. I have tried different ligation ratios (1-20) but NO colonies.

 

ANYONE WITH SUGGESTIONS THAT CAN HELP ME GET PASS THIS STEP?



#17 bob1

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Posted 04 May 2018 - 06:03 AM

In response to your previous post:

 

I would say that you have one enzyme that is cutting completely - this is good, it's what you want to happen.  I say this because plasmid preps usually contain multiple forms of the plasmid in different energy configurations, such as supercoiled, circular, linear, nicked etc. Your prep appears to have most of the DNA in one form, this is most likely circular (it's running small), which means that you did just fine in your prep, but you should pay strict attention to the time for alkaline lysis. If you do this you will get some super-coiled out too.  Your concentrations are a little low, but not too bad for a beginner.

 

Your reaction conditions are good - you have at least a 2x excess of enzyme over DNA. 

 

However, the other enzyme is not cutting - most likely this is the XhoI which works less well in buffer O. One good way to get around this is to do sequential digests - do one digest, clean up the DNA by precipitation, then do the next digest in the recommended buffer for that enzyme. To do this it is a good idea to start with a lot of DNA (maybe a ug) and work from there. Don't scale up the volumes for the digest, just make multiple tubes of the digest reaction. 

 

If your enzymes have been stored correctly and kept cold (i.e. only taken out of the freezer just as you are adding it to your reaction) then they should last more or less forever. I have (admittedly 5 years ago) used enzymes that expired in the 1990's with no problems.

 

Which ladder are you using for your gels? Knowing the band sizes is half the battle in working out what might be going wrong.



#18 bob1

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Posted 04 May 2018 - 06:18 AM

 I have a codon-optimized gene cloned into pUC57 with AvrII and XmnI overhangs. I want to subclone the gene into a yeast expression vector (pKOV410). The size of my gene is 1429 bp and the expression vector is 6352 bp. The overhangs are close to each other on the vector. I did a doble digest on the pUC57 to release my gene, ran a gel and extract my gene of interest using Promega gel extraction kit with a final elution volume of 50 ul. I get 20ng/ul concentration of my gene. I also did a double digest of the pKOV410, run and extract the linearised vector. The nanodrop concetration of the vector is 45ng/ul. I am using NEB ligation kit. I tried ligation at different environment (3hr RT, 16hr O/N, 4 degrees O/N). I take at least 3-5 ul of my ligation into 50ul JM109 competent cells which we just bought. I do a control with uncut vector and i get hundreds to thousands of colonies. The ligation kit is new and i made aliquots of the buffer the day i received the kit. The enzymes are from NEB & they recommend digestion for 5-15min, but it seams not to be enough, so i went for 1-3 hours, that works well. But i still dont get a single colony. One of the overhangs is blunt & the other is sticky. I have not tried sequential digest yet. The antibiotic resistance for the expression vector is Kanamycin. I have tried different ligation ratios (1-20) but NO colonies.

 

ANYONE WITH SUGGESTIONS THAT CAN HELP ME GET PASS THIS STEP?

OK. First things first - you know your digest worked on pUC57, but did it work on the pKOV410?

Are you growing the pUC57 yourself or did you get it straight from a vendor? If from the vendor, check the sequence, perhaps it doesn't grow well.

 

Make sure you are growing at 30C for the pKOV under Chloramphenicol selection (At least for pKOV strains according to Addgene - but check your product sheet)

 

Blunt ligations are inefficient, overnight at room-temp should work. If you are still struggling, try adding some PEG8000 as a crowding reagent.

Try adding some more ATP to your reaction - it easily degrades even when stored properly.

 

It is counter-intuitive, but ligation reaction components can be inhibitory to transformation - try transforming 0.5, 1, 2, 3 ul of the reaction and plating.



#19 Mashudu

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Posted 05 May 2018 - 01:59 AM

Upon receiving the pUC57, I transformed and grew the pUC57 and made glycerol stocks so that i do not run out of DNA. I am using the glycerol stocks in my experiments. I have been growing pKOV at 37C. What about the fact that the two ends are close to each other? Would you suggest that i do a sequential digest?



#20 bob1

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Posted 05 May 2018 - 12:34 PM

It might be a problem, but so long as they are more than about 3 bp apart, it should be fine, so long as cutting with one doesn't disrupt the recognition sequence for the other. I see that XmnI has a large recognition site, so if you were to do a double digest, I would do that one first.







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