I do Cloning:
in ligation: 15 µ DNA
5 µ Plasmid (pks), 2/5 µ H2O, 0/5 T4, Buffer 2µ Added and 90 Minute at a temperature of 16 ° C gave.
The resulting product is then transferred into competent cells using heat shock.
Finally after 2 hours, bacteria grown in medium containing ampicillin was culture.But finally, after only a few colonies were grown for 24 hours.
Which unfortunately with the PCR colony I did not get any results.The colonies were grown without the gene.I've reviewed several stages of his career, but because it's difficult to not catch.
I would suggest that you do a miniprep and look at the pKS on an agarose gel. See what size it is. The molecular size of the vector will tell you whether there has been ligation or not. Since there are colonies I am assuming there is no problem in transformation. In case you have all re-circularized vectors, clearly there is a problem with ligation. What about your insert DNA, is it a PCR product, and does it have the right sticky ends. You could try either by dephosphorylating the vector, or by using two different restriction enzymes, that is clone through a double digest.
Hi Mr Vivek
I've used two restriction enzyme XbaI and SacI before the Ligation.Once a colony of PCR could take a single clone containing the desired fragment. But,I then did a plasmid extraction and again extraction of the PCR product of a negative result and could not put the fragment see.!!!!
I do not understand...you have done a double digest followed by ligation. In principle then you cannot have recircularization of the native vector. So, what are you getting out of the transformed cell? A linearized plasmid DNA will not replicate.
In case you are confident of your insert DNA, and the vector, you need to change the enzymes and see, also, ligation is very sensitive to ATP. Take a fresh stock of the ligase, and all ingredients...
I agree with Arjan. It is not generally recommended to add ampicillin during the recovery phase following transformation, although beta-lactamase does get expressed within minutes of transformation.
Please note that both SacI and XbaI are not the easiest restriction enzymes to work with. SacI is very sensitive to inhibition by salts, so if your DNA quality from the minipreps is not good, your vector will not cut. In addition, XbaI is blocked by dam methylation. If your insert has the sequence "GA" preceding your XbaI site, it will not get cut if you are using a dam methylase positive strain of E. coli. If your colony PCR is positive, but your restriction digest is not, you should sequence the plasmid to test whether an insert is present or not (or use a different set of restriction enzymes, if possible).
Hi
I am already a plasmid gene sequence is a promoter into it. I must put their genes Continue on with this promoter.That's why I do not have any other option except ampicillin to select colonies. also ,Except this two enzymes can not use the other enzymes. E. coli strain used is DH5α.
Your comment could not act on the enzyme T4. Or as little time Ligation at 16 °?.
I'm sorry but the way your reply is written, it is impossible to understand what you are talking about. T4 ligase can work at room temperature or at 16C. No one is suggesting that you do not use ampicillin, but you must wait at least 5 minutes post-transformation, before introducing the antibiotic. And the antibiotic should be on an LB plate not in liquid culture, until after colonies have grown.
DH5a is methylation competent. Check your sequence to see if you have a GATC methylation site flanking your XbaI site in the recombinant (with cDNA insert) plasmid before proceeding with your digest.
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