Cloning pCAEGFP into XL1 E. coli
Posted 18 November 2004 - 02:49 PM
I am trying to clone an insert (1025bp) called N302X into a 7000bp vector pCAEGFP. This vector has the gene to Kana resistance. Well I digest both DNAs with 2 enzymes and remove the bands from the gel. Till here everything is OK and I get really good purified DNA.
Well I want to join both with T4 DNA ligase from Biolabs and its 10x buffer and incubate for 1h at RT. I electroporate it and them try to clone it in XL1 in KANAr plates overnight.
For a month and I get abs no colonies!!!
I have even tried to use different T4 DNA ligases and the result is always the same! NO COLONIES!!
Please help or give me some advise!
Posted 22 November 2004 - 03:38 PM
Some steps to check would be:
1) The efficiesncy of your restriction digest: are you digesting your insert out of another vector, or is it PCR? What about the vector itsef? Basically, are you sure you are getting the fully digested product?
2) Run a sample of ligated product on gel, check that you are actually getting ligation happening
3) test your bugs with another vector (+ve control) for ability to get transformed
4) check the concenration of kana in your plates, with a different bug say.
Depending on these results you could figure out your problem. Good luck!
Posted 23 November 2004 - 02:23 AM
With the previous recomendations, I would check also:
-Your competent bacteria. Try with another vector to test if they are really competent.
-Try to obtain colonies with different Kama concentrations, try with concentrations below the usual ones.
The way is to put a control in all your steps to be have an idea about where is the problem