Hi,
I am stuck with a cloning experiment:
The insert & backbone(bb) share one compatible end at 3' end(NotI) and one incompatible end at 5' end (XbaI for insert; HindIII for backbone). So i first digest the incompatible end and blunt it with Klenow Pol. I and then proceed with digestion of the compatible ends before ligating with T4 ligase at 16°C o/n. I did this a bunch of times already and received many colonies for undigested ctrl, a hand full for backbone-only ctrl.. No colonies for bb+insert.
I also tried a pcr approach: amplifying the insert and adding a HindIII restr. site. Primers were designed after sequencing. Here the amplicon sizes as well as digested amplicon sizes are completely off.
Can you think of any (uncommon) alternatives?
Thanks in advance!
Use a Topo-TA cloning kit. As long as you have an A overhang (added by A-tailing your PCR product), you can clone in any insert.
Hi Anton, I would suggest you to try this approach (find attached the paper), it is quite flexible and I have tried using a different strain of E.coli (DH5a) instead of XLBlue and it works perfectly. Also, I have used a shorter homologous ends (around 15-18 bp and no problems).
Are you cloning into an E.coli expression vector? Consider that the protein could be toxic for E.coli and that is why you are not getting any positive colonies.
Hi Katie & Jiang,
thank you so much for your suggestions! i will get into both methods.
@Jiang I used Top10 & DH5a, neither worked. The protein shouldn't be toxic as i get the original plasmid from Top10. My aim is to clone the insert into another backbone.
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