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Sandy143

Member Since 19 Feb 2009
Offline Last Active May 06 2013 08:34 PM

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Parasitology, molecular medicine

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2xzwei
24 Apr 2013 - 08:39
Epigeneticist
05 Feb 2013 - 18:00
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Posts I've Made

In Topic: Problem with double digestion: one enzyme is not working

21 April 2013 - 11:03 PM

its something new for me..thanks a lot memari

In Topic: Problem with double digestion: one enzyme is not working

19 April 2013 - 01:03 AM

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.

In Topic: Problem with double digestion: one enzyme is not working

17 April 2013 - 07:55 PM

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

In Topic: Problem with double digestion: one enzyme is not working

16 April 2013 - 07:45 PM

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.

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