So I have been trying to make a construct. But I am seeing no insertion of my desired insert into the vector. So let me tell you guys some details
1. I am doing double digestion of my pET3a plasmid and my insert with Nde1 and BamH1-HF enzymes using cutsmart buffer. And then for both the cases, am gel purifying it.
2. For ligation, I am using quickligase, and have tried concentration ratios of 3:1, 5:1, for insert:vector. i also tried incubation times like 5mins, 15mins at room temperature.
3. I am transforming in E.cloni-10G cells using heat shock and am getting colonies on Amp resistant plates.
However, every time during colony PCR I am seeing no desired band, so the colonies I am observing are all having empty plasmid.
I did several controls, like doing single digestion with either of the enzyme and gel shows that they have been linearized everytime. So I thought the enzymes are good. Although for BamH1 the band is broadened.
I have been done controls during ligation step, like without the enzyme one time, or without the insert. But whatever colony I am seeing is of the empty plasmid.
Can someone advise me?
Thanks
Kushal
hi, i just saw ur whole procedure, I think ur transformation is fine. bcoz ur bacteria had the resistance, right? so I think maybe u can focus on the ligation step. Maybe u can run the electrophoresis gel after ur ligation step. if the insert group didn't have any difference compared to ur plasmid only group. it seems that the problem is ur ligation step. regards.
1. Please do the sequential digestion so that you can ensure the proper digestion of double digetsion.
2. Check the wheather the enzymes are working or not by doing single digestion of pET3a and the same you can use for sequential digestion.
3. You are getting all negative colonies because mosltly due to self ligation.
I agree with Kanduri, always avoid double digests. (i) Go sequential and check your digests by electrophoresis. Sounds old fashioned but it works. (ii) Secondly for the ligation, you use different ratios of insert to vector. This is good, yet make sure you calculate those ratios on a molar bases, not on weight. Very often the molar weight of your insert will be very different of that of the vector. (iii) if you have a unique restriction site on the vector between the NdeI and BamHI which is not present in your insert, use that restriction enzyme to cut your ligation mix. Any self closing vector will be cut up.
Thanks for the suggestions.
So, my transformation is fine as i have done controls with just empty vector and my previously made construct plasmid. Those work.
As for restriction enzymes, I know they are working fine as I tried to do single digestion with both and gel shows that the the plasmids have been linearized in both the case.
However, I have couple of doubts my self. Do you think that I need to do PCR clean up after PCR amplification of inserts, i.e, before double digestion.
Also, after digestions is there any other way of cleaning up rather than gel purification?
Regards
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