After I extract the DNA using powerlyzer kit and then then PCR it then using QIAquick gel extraction kit (250) to purifiy the PCR then there was a problem my sample smelt ethanol I used vacuum centrefuge at medium temp. For 30 min then re suspend the pellet by adding 25 microL of deionized water. I have checked on gel (using 3micro L of my PCR product) and shows that I have purified PCR product on the gel then I did the ligation using invetrogentopo ta kit I follow the protocol all the way through (the pulse was 1.8v for 3.9sec) till the plating on LB +kanamysin (50microl/ml + xgel 80microL/100 ml) but I did not get the colonies while other are using the same method and they have got the white and blue colonies please if anyone have any idea how I can solve the problem? Thank you.
Have you made sure the polymerase you are using for your PCR has no exonuclease activity? If there is exonuclease activity you won't have 'A' overhangs on your PCR product and therefore won't be able to ligate your fragment into the vector using this kit.
Dear A. Van, sequencing is not influenced if you use any polymerase for PCR. Now coming to your problem, i think either your X-gal solution is old or you require IPTG induction for blue color appearance in your E.coli strain. Make plates for LB + Kan and spread IPTG + X-gal solution over it and let DMF evaporate in laminar hood. Now plate your transformed cells and after O/N growth keep plate at 4' C for 1-2 hrs. Hopefully blue color will appear.
Ari Van, you work in dim light while handling X-gal as it is light sensitive.store in brown eppendorff tubes. Follow everything as given in sambrook et al. it will work for sure....
Just Beginner
thank you ,the Xgel is in dark tube plus fully foiled all around i do not think that was a case as i have borrowed couple a plate from a PostDOC who have got white and blue colonies and the kit was working for him...but i did not get any...
thank you for ur comment
Dear Loi Vu
no i have not thought about that to be honest but today i have tried to use pGem T kit from promega. see what happen .. in case if i didnt get any i will go back to use invetrogen kit again. and seek info from all of you guys
thank you for your comments and advice.
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