ligation again - (May/15/2004 )
I plan to insert 1.5 kbp , 500bp and 1150bp fragments into aav(6.1kbp) separately.
I inserted the fragments in TA clone and cut down by ECOR1 and BamH1. and purified by Qiagen gel purification kit(water dissolved). The vector was treated in the same way. I ligated the fragments and vectors (3:1)as the invetrogen recomend condition (t4 1ul) at 14.C overnight. But nothing grew up(even the digested empty vector did not grow) My competent cells are ok and ligase still works. I also tried to purify the digestion products by ethanol after gel purification and ligated series ratios. I douted if the two enzyme sites are too close(6bp),to be cut inefficiently? One cutting and two cuttings look the same in the gel. It seems that I have tried all means but still failed. Can any one give me some suggestion?
it may not be due to the inefficient digestion of your empty vector since the digested empty vector also didn't grow. if you worry about this, you may digest the vector overnight using Biolab or other good enzymes. gel-check both insert and digested empty vector after purification, and the ratio could up to 10 or more. i had met same thing, but the vector is 13kb which is hard to be ligated. and i increased amount of digested vector to ~300ng and it's ok. by the way, you can heat the mix of insert and vector at 60centigrate for 5min and cooling on ice for several miniutes, then add buffer and ligase.
Don't worry, always there's a solution.
First, try to digest your plasmid and runa a agarosa gel to check if is already cut. Then purify from gel (with Quiagen or Geneclean), later with SAP(Shrimp Alkaline phospatase from Amersham) desphosporyle your plasmid to make sure that can not religate alone (without your fragment).1 h at 37ºC it's ok, and inactivate SAP 15 minutes at 65ºC. Then purify the plasmid from solution with one kit(QUia or Geneclean).
Be sure that your cells are competent(MAKE A CONTROL: transform 1 ng of a plasmid with insert in 100 microl. of competent cells, DH5alfa are great!!)and see the eficiency of your cells.
Later when you make the ligation try 2 or 3 ligations with diferent rates, like 1:3, 1:4 and 1:6 and make a negative control where only put your plasmid without insert (then you can see the eficay of the SAP to avoid religation of the plasmid alone)
Another thing that you can do is: if your not interesting in the direction of your subcloning don't cut with 2 RE, try to make blunt ends. To make it, you first cut with one RE and later make blunt ends with Klenow (Amersham) 30-60 minutes at RT, purify and make the same with the plasmid but also treat with SAP.
I'll hope I'll help you. Good luck!!
hi, did you fix your problem by now? i think the problem is Vector digestion. your vector only digested by BamH1 or EcoR1, then, all the vector ligased with the insert, that can explain why you cannot get anything from transformation. if you fix the problem, please let me know, i was nearly mad for the same problem