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Problem with double digestion: one enzyme is not working - (Apr/15/2013 )

Hi there, I used to digest pET28 vector and my insert sized 700bp with BamHI and HindIII prior to ligation. The insert was amplified by PCR using specific primers with the respective RE. However, sequence result showed that the target gene was inserted to the vector at location of HindIII only (the insert carries ~ 100bp of TOPO sequence). I wonder why the BamHI is not working? I use BamHI buffer and all enzymes are from Fermentas. Many thanks

-Sandy143-

What makes you certain that the BamH1 isn't working - did you do a single digest control to confirm this?

-bob1-

I did double digestion and after ligation into pET28 and transformation in TOP10, straight away I send the plasmid for sequencing (I did PCR colony to confirm the good clones). The F primers was designed as BamHI-target gene and R primer as this: target gene-HindIII. However, from the sequence result I found that my insert has additional TOPO sequence in front of BamHI location, around 100bp (with HindIII site within) and the insert was incorporate into pET28 at HindIII sites. The sequence is roughly like this: pET28 sequence (stop at HindIII) - TOPO sequence - target gene - pET sequence. For troubleshooting, I did check the BamHI enzyme I used before by doing single digestion, and it seems okay. I was thinking to do sequential digestion in which I can ensure BamHI is working well but this has made me wonder why it happened. Many thanks for your help.

-Sandy143-

So where did the topo sequence come from if it isn't supposed to be in pET28? It seems that you probably cloned into a non-empty vector.

-bob1-

Thanks for your reply. yes I agree with you. Do you think that sequential digestion can solve this problem? Thanks

-Sandy143-

It should work fine as a sequential digest, probably best to clean up between digests and use the recommended buffer for the each enzyme.

It is quite likely that the PCR product has not digested well as this is often the problem with these sorts of clonings. Make sure that you added 6 bp of sequence 5' of the restriction site to ensure that the RE has somewhere to bind.

Also note that for primers, any additions like restriction sites need to be put on the 5' end of the primer, so as to ensure that they are outside the region of interest.

-bob1-

Yes..for the primer I'm pretty sure I've done all that. I also did the sequential digest, waiting for the sequencing results now. If it still doesn't work, I think I will redesign the primer with another RE.

-Sandy143-

Did you consider this

https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments

you can use this new buffer too:
http://www.nebcutsmart.com

-memari-

its something new for me..thanks a lot memari :)

-Sandy143-