I cut pcmv6-AC-ha-his(origene) with hind111 and xho1, the same as my insert whose size is about 1k. Then I ligate them which labeled as "target" ,and negative control is the ligation system which include vector, ddH20 and ligase. The result is as follows: the negative control grows no colony, but the "target" grows many colonies(about 3000). I pick up 16 colonies, but any colony is not right. All materials are same between negative control group and "target" group.
Given that your vector only control showed no colonies, in principle, all your 16 picked clones should have your insert. But this not the case. I suggest you the following troubleshoot strategies:
1. Dephosphorylate the vector backbone (pcmv6-AC-ha-his) to minimise the false positives resulting from the self-ligation of single cut vector.
2. If your insert is a PCR product, it is necessary to add some overhang sequence (~10-bps) upstream of the restriction sites HindIII and XhoI for an effective digestion.
3. Include also insert only control. This should NOT give any transformants. This confirms that your insert is clean.
Hope this helps!
All the best,
Manjunatha
Dear Yuanyuan,
Please let us know what you mean exactly by saying '' but any colony is not right ''...
Did you perform colony PCR? and didnt you get any desired band?
And also how did you prepare your gene of interest? did you do the gel extraction of your gene?
@ Saber, she means none of the 16 colonies had the insert.
@Yuanyuan, the fact that your negative control did not get any colonies is alarming. You should get some colonies in the negative control, even though you digested with HindIII and XhoI.
1- Try to CIP your vector and do the ligation again.
2-Adjust the vector to insert ratio. How much insert and how much vector do you use in the ligation reaction.
3- Make sure your ligation is working by running some of the ligation reaction on agarose gel and see if the insert shows higher molecular weight supercoiled bands.
4- Gel purify your vector and insert if you have not done so already.
You have to give more details to help you troubleshoot.
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