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Genomic library DIFFICULTIES!

puc19 lipase gene no RE digest stressed

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#1 LTSAL

LTSAL

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Posted 18 June 2015 - 02:46 AM

Dear researchers please help

 

Im actual;y working on cold active lipases. Currently trying to look for novel lipases. Here is what i did:

 

I did intial digest with Sau3A1 with my genomic DNA with different concentration and mange to get a band of 10kb to 2 kb in which the lipase genes are believed to be in.

 

I ligated my product into puc 19 (vector) and host of DH5 alpha. I seem to get good no of colonies on the plate however when I test with RE digest (double digest) i just get a smearing band. Im using Eco R1 with Hind111 buffer tango

 

Please help. Attached is the gel with harvested plasmid. All of them seem to have  a band which is above 10 kbIM000530 (new).jpg

 

Please help!!! Do i change the plasmid, or the RE?







Also tagged with one or more of these keywords: puc19, lipase gene, no RE digest, stressed

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