long insert ligation - (Feb/17/2008 )
Hi everybody
I try to ligase my insert (1764 bp) into pET32b+ vector which cut by NdeI and XhoI enzyme. But i can not get any colony after tranformation. Any ploblems which occur in my experiment? Please share yours idea. Thanks. 
I try to ligase my insert (1764 bp) into pET32b+ vector which cut by NdeI and XhoI enzyme. But i can not get any colony after tranformation. Any ploblems which occur in my experiment? Please share yours idea. Thanks.
Many reasons for 'no colony'. Anyway, your insert (1764bp) is not long.
I recommend you to make negative control (vector) in ligation. This solves many problems in ligation.
1. Probably vector plasmids were degraded. Prepare new plasmids with new buffers.
2 Probably the concentration of your antibiotics in your plate(or even antibiotic itself) is wrong.
Any control for the competence of your cells?
Please, give us a bit more details on your protocol, as for now it's a bit too vague to really help you out (have you checked the activity of your restriction enzymes, how have you purified your vector/insert after restriction, was the ligation buffer thawed often before, ...).
Look at each step of cloning carefully, just to make sure nothing has gone wrong especially the buffers or solutions used for cloning.
How much DNA was used for ligation?
the NdeI is difficult to ligate because it need ligation of blunt end, so improve the amount of ligase and the insert concentration
I try to ligase my insert (1764 bp) into pET32b+ vector which cut by NdeI and XhoI enzyme. But i can not get any colony after tranformation. Any ploblems which occur in my experiment? Please share yours idea. Thanks.
Be sure both enzymes are cutting using controls.
Is your insert a PCR producto or a product of digestion with same enzymes from other plasmid?? If it’s the first case and the cut sites are in the oligos, enzymes may are not cutting because they are too near from the ends, you sould clon it fist in a T vector or something.
Have your cells good competence?? Use a positive control (0.1 pg of plasmid, for example).
Use a positive control for ligase too (a plasmid digested with one enzyme and religated).
Use a background control too (same plasmid digested without adding ligase).
Se sure your ligase buffer has not been frozen-defrozen many times, if it’s the case, you could increase ligation adding ATP.
Let the ligation reaction over night at 4C or 14-16C.
Do the ligation just before digesting and purifying bands. Although, they could work after month of digested, using “fresh” purify band work much better.
Hope this tips could help!
Cheers!!