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Selfligation of Novagen pET-42b is not working - (Mar/15/2006 )


I tried to ligate an oligo of 130 bp into pET-42b and never received any clone. Step by step I found out, that the selfligation of the once cutted (Nco1) vector is also not working. This ligation should be "highly efficient".
I have absolutely no clue what can be the reason.
I took the plasmid directly from a miniprep (qiagen) and digest ~ 1,2 µg (30 µl) with 1 µl (10 units) Nco1 @ 37 °C for 2 h. After this step I accomplish a gel purification (qiagen). From the gel I know, that the plasmid is fully digested. From the digested and purified pET-42b I prepared a dilution series to estimate the concentration using agarose gel electrophoresis. I received ~16 ng/µl digested and purified vector. I used 5 µl of this and 1 µl of T4 Ligase (NEB) in an end volume of 20 µl and left it for 16 h @ 16 °C.
The transformation was done with Nova Blue competent cells (Novagen) like described in the protocol (20 µl of cells + 1µl of ligation mixture 30 sec @ 42 °C + outgrowth and plating). With the positive control (1 µl of undigested pET-42b + 20 µl competent cells) I receive a plate full of colonies.

Any suggestions? blink.gif



UV damage when you cut out the gel band is a possible cause. UV-generated damage to DNA stained with ethidium bromide reduces the transformation efficiency. BioTechniques 1999, 11:747-748 and BioTechniques 1996, 21:898-903 address this problem. One minute of exposure to 312 nm UV can reduce the transformation efficiency by > 90%. (BioTechniques 1999, 11:747-748)

UV-damaged DNA products include cyclobutane pyrimidine dimer (T-T), 5-thyminyl-5,6-dihydrothymine, 4,6-diamino-5-formamidopyrimidine, 5-hydroxy-5,6-dihydrothymine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 6-hydroxy-5,6-dihydrocytosine and 8,8-adenine dehydrodimers (from poly(dA). Exposure of DNA to UV light prior to transformation resulted in 2 e 4 cfu/µg compared with 1.7 e 6/µg when the DNA was exposed to a Dark Reader, an 85-fold reduction in transformation efficiency. (H.A. Daum, III and M. Seville, 2002, Cutting back on UV damage. Modern Drug Discovery , Jan, 02, 18-24.)