g***m
发帖数: 465 | 1 man, refer to gzip's post.
the ligation is somehow vulnerable.
1. check with your lab fellow to make sure your ligase is still alive.
2. always use latest ligation buffer, you can even smell the thio stuff if you
are using NEB enzyme.
3. critical thing is your insert: never directly use the insert purified from
gel, you need gel purification followed by ethanol precipitation to make sure
the purity. However, you can use gel extracted vector for ligation in small
volume.
4. add enough insert, for | M****e
发帖数: 70 | 2 with my experience, the suggestion is to check the PCR product
first. i would rather not digest the PCR product, actually, it
is better if you can subclone it first. for double digest with
NheI and XhoI, if you are using NEB buffer, use buffer2. digest
the insert and vector for enough time, and make sure your enzymes
are good (i think so from your description). unless the cloned
gene is harmful to the bacteria host, the cloning problem often
come from incomplete digest for such cloning strategy. |
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