6 replies to this topic

[peixumol]

Hi,eyeryone!I'm keeping cloning a 1.5kb pcr product using a TA cloning system,but most of the white clonies I checked just contain some smll bands rather than the expected 1.5kb one. who experienced friends will give me some guides?

[DevGrp]

Two questions:
Does your PCR also give some primer dimers?
Did you run your PCR product on a gel and then cut out the band you want, purify it and then cloned it?


I have experenced similar problems and put it down to cloning the primer dimers / small unwanted PCR products. Gel purifying the band you actually want to clone first. solved it for me.

Hope this helps
Ian

[Daniel5306]

Are there possibility that your 1.5kb sequence contained the restrict enzyme site using of that enzyme clone your sequence into the vector? Of course, if you have purified your sequence before the ligation, that is not the problem.

[peixumol]

hi,thank you for all  your suggestions! but I'm still puzzeld now,because I've  really cut the 1.5kb band and purified it and confirmed the successful recovery . so are there more possible reseasons for this? I'm really appreciated!

[phaffia]

Hi

was just wondering how you've checked for the presence of your insert, PCR? or restriction digest?

Thanks

[peixumol]

Hi,ffafia, I checked by pcr,with the M13F/R universal primers.any suggestions?

[phaffia]

Was thinking it'll be worth trying a restriction digest to check for your insert. If your PCR is not optimised, there might be a chance that you are not amplifying your insert at all, hence producing a small pcr fragment. Did  your positive control turn out well in your PCR reactions?

You should be able to tell which clone contains your insert (by restriction digest) since 1.5kb is a pretty big insert.

Hope this helps.

Cheers, phaf

Edited by phaffia, 10 December 2004 - 12:56 AM.