ligation - the problem of ligation? (Jun/12/2002 )
Part of substracted products(5ng/ul) 1ul was ligated with 1ug xhoI-digested ,calf intestinal phosphatase-treated pBluescripts(sk) overnight at 14c and transformed into DH-5a bacteria.But there were no white single clone.I want to know the reason .Could you help me?
Use the low quantity of the subtractive sample,for example,0.7ul.Because the salts in the PCR sample may inhibit the ligase.
But the PCR sample was purified ,and the problem of salts will not exist. Whether the low quantity of the substracted products(5ng)used in the ligation affects the results?
You must absolutely perform controls for background i.e. CIP treated vector + ligase without insert. It seems that 1ug vector is too high. Depending on the ligation reaction volume your vector conc. may be favoring intramolecular interactions instead of intermolecular interactions. The vector conc. should be no more than 5-10ng/ul in the final ligation reaction. Also try to increase the amount of insert to about 1:1 if you can calculate based on the average size of cDNA.
(Edited by milanoj at 2:13 am on June 13, 2001)
Whether Lowering the quantity of vector will not ligate the fragments which express low in the plant ?