Double digestion of PCR product and ligation - (Aug/11/2005 )

HI, sorry for my english...but if you understand...Help me!

I have some problem with double digestion EcoRI/XhoI in PCR product.

My fragmet is short 600 bp and plasmid is 10000 bp.
I'm sure that double digestion of plasmid occurs ( I have use as blank plasmid with insert of 400pb and it was excise and I see my plasmid linearizate) but for PCR product I can't have any...

I have those primers:
primer foward GgaattcATGAAG.... (gaattc: EcoRI)
primer revers CCGctcgagCTAGTC... (ctcgag: XhoI)

It's possible that I have little bp near restriction site but... I can't order new primers!

Ps: today, when I see my ligation... there are any Ecoli cells!!! Then, I think that digestion of plasmid was right, but none for PCR product...

I have no more time!

Thanks.

I think you will have trouble cutting with EcoRI as you have only one base extra on the end. If you can't order new primers then phosphorylate your primers, make them blunt ended with klenow, then ligate them together into a long multiple PCR product fragment (should be over 10kB). Then you can digest with your two RE and you will know that the PCR product is digested correctly.

Daniel

Longer automated DNA sequencing reads

[quote=Daniel Tillett,Aug 11 2005, 12:04 PM]
I think you will have trouble cutting with EcoRI as you have only one base extra on the end. If you can't order new primers then phosphorylate your primers, make them blunt ended with klenow, then ligate them together into a long multiple PCR product fragment (should be over 10kB). Then you can digest with your two RE and you will know that the PCR product is digested correctly.

Daniel

Thanks for your reply, but I think that you tell me not sound good...
because when I have a long multiple PCR product fragment, who tells me that I have EcoRi-fragment-XhoI-EcoRI-fragment-XhoI-XhoI-tnemgarf-EcoRI.... and when I digests (better: EcoRi and XhoI digests!), I can have EcoRI-fragment-XhoI-EcoI.... and many others... and when I ligate many colonies have a no good plasmid with insert.

Thanks.
Marco.

After you digest the concatenated PCR products digestion with both RE this will generate the correct product because you will be cutting the correct 600bp fragments out of the middle large product. It does not matter what order the PCR products ligate together.

Because you have so little extra bases on the end then it is possible that some of the EcoRI-EcoRI site will not both cut. If this happens then only half your clones will be correct. This shouldn't matter as it will then just be a matter of screening your clones.

Daniel

DNA sequencing reagents

[quote=Daniel Tillett,Aug 11 2005, 02:01 PM]
After you digest the concatenated PCR products digestion with both RE this will generate the correct product because you will be cutting the correct 600bp fragments out of the middle large product. It does not matter what order the PCR products ligate together.

Because you have so little extra bases on the end then it is possible that some of the EcoRI-EcoRI site will not both cut. If this happens then only half your clones will be correct. This shouldn't matter as it will then just be a matter of screening your clones.

Daniel

Daniel,
Thanks for your help, your idea it's good, but I'm not sure that I'll use because you forget that some site will be not cutting indipendent of my condition. Then I can Have a "false" good fragment contain: (EcoRI-Fragment-XhoI-XhoI) or (EcoRI-EcoRI-fragment-XhoI) or (EcoRI-EcoRI-fragment-XhoI-XhoI). MORE THAN half of my clones will be uncorrect no?
Yours sincerely.
Marco.

Yes half your clone will be incorrect, although it is always better to have 50% correct clones than none at all

Another alternative is to order two short (~12bp) complementary oligos that can form a short blunt end dsDNA fragment (adaptor). PNK the oligos and ligate them to the end of your PCR fragments and then digest with your two RE.

Daniel

DNA sequencing analysis programs