I plan to digest the whole bacterial genomic (klebsiella pneumoniae) by EcoRI or HindIII, and then ligation it to the same site of vector pBK-CMV. The question is should I purified the digest before ligation? If I purified the digest, the length of my target fragment is unknown, is there any kit like PCR purified kit can purify the digest?
Thank you very much!
Hi
You can straight away inactivate the EcoRI and HindIII after digestion and proceed for ligation. These two enzymes have been worked out very well and there buffers are also compatible with ligases (but still check the literature). If you purify the ligation will be more efficient.....The commercial column based kits will give less yield as your fragment size increases....I'll suggest you go for regular phenol:chloroform purifications followed by precipitation with sodium acetate and ethanol...it works out very well.....if you are concerned with recovery of smaller fragments..include carrier molecule like glycogen or LPA....
bye
there is no need, You can straight away go for the digestion.. unless the level of contaminants is too high..like with proteins.. then phenol:chloroform separation will do...
Thank you for your valuable advice, Ajay Saini and Sudheesh Ap, I 'll continue my experiment tomorrow, and keep in mind of your suggestion, thank you again!
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