I'm trying to insert a ~5 kb PCR product into a pJET cloning vector which is ~3 kb using blunt-end cloning.
I am able to amplify the amplicon successfully, then I perform the ligation of the amplicon into the cloning vector, transform the construct into DH5 alpha cells through heat shock, culture on LB with an ampicillin selection marker, culture in liquid LB+amp, extract the plasmid DNA, and finally perform a restriction digest using Bsa I and Spe I, expecting two bands at 1.6 kb and 6.3 kb.
Despite all my efforts and attempts to tweak the protocol (adjusting volumes, changing the ligation time from 1 hr at 22 C to 16 C overnight, etc etc etc), I ALWAYS end up with a band around 3 kb - 3.5 kb.
Sometimes, two bands around 1.3 kb and 1.7 kb would appear in addition to the mysterious 3-3.5 kb band.
Anyone have any advice for making such a large construct? The pJET vector has previously worked for much smaller constructs (around 6 kb and below)
Which enzyme are you using?
That would be the initial step and it would assist in order to answer your question and give clear protocol
For amplifying the 5 kb insert, I used Primestar GXL DNA polymerase (denaturation at 98 C for 10 s, anneal at 60 for 15 s, extend at 68 for 4 mins, 33 tot cycles). Afterwards, I performed a ligation into the pJET vector using a T4 DNA ligase (16 C for ~20 hours; also previously tried it at 22 C for 1 hour).
Added the diluted ligation product to the competent cells and heat shocked (10 min on ice, 45 s in a 42 C water bath, 3 mins on ice).
Plated the cells on LB+amp (100 ug/mL), took colonies the day after, and made overnight liquid cultures.
Extracted DNA using alkaline lysis, then performed BsaI and SpeI digest at 37 C for 1 hr.
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