Cloning - Bacterial gDNA in minipreps? (Jun/14/2007 )
I am trying to clone a 25S cDNA fragment (900 bp) into pGEM-T easy. I have done the ligation (overnight) and transformed XL1-Blue cells and plated on LB/Carbenicillan plates. I've then miniprepped with a Qiagen kit and cut with EcoRI to drop out inserts from selected preps. I am getting unexpected band sizes on my gel, such as a 2.5 kb band and a very high molecular weight band, with a very faint approximately 4-5 kb band in between. I have done several concurrent ligations/transformations with the same vector, cells, and plates and they all have worked fine. I have repeated the 25S ligation/transformation a couple of times and get the same weird banding pattern. I thought it might be bacterial genomic DNA coming through the miniprep, but I don't know. Any suggestions?
Hi Posting a picture would help, but my guess is that it is incomplete digestion of your plasmid.. Check out whether your construct is methylated as EcoR1 is methylation sensitive and supercoil sensitive.
well when it's enomic DNA contamination, you have a smear as genomic DNA is fragmented.
when you have a specific band, it's rather sthg you don't want in your DNA rather than having genomic contamination.
Most often it comes from the template of your PCR (if your insert comes from a PCR) even if you digest ang gel purify your insert.
when you have a specific band, it's rather sthg you don't want in your DNA rather than having genomic contamination.
Most often it comes from the template of your PCR (if your insert comes from a PCR) even if you digest ang gel purify your insert.
hi..
It can't be a Genomic DNA...
The basic principle of miniprep is all the genomic DNA should be precipitated in centrifuge as its always attached to the membrane. So you should not see any genomic DNA..
But if you have.. then it can't be 4-5 kb.. as Ecoli Genome size is 4,639,221 (4,63kb). If will never run in gel.. if its there in gel.. then u would see it in to loading chamber.. so don't worry about that...
problem is in your PCRs. It could be your main PCR or Colony PCR.. check your primers.. you are getting some unspecific priming..
I always use PGEM-T which is a perfect system...
what are your controls?
Thanks to everyone who replied to my questions. Since my post, I have found that the plasmid/clones will cut with two other flanking primers in the multi-cloning site, so there is an insert and the clones won't cut with EcoRI. The EcoRI enzyme is good because it cut many other concurrent clones fine. So, it looks like some inhibition of the EcoRI enzyme. I'm not sure exactly how methylation works, but the cells I used for transformation are XL1 Blue and I don't think they prevent methylation. Also, the partial gene sequence from a closely related species that I based my primers on for this clone is high in CG's and CNG's. So would you conclude that methylation has occured? How do I check out whether it is methylated?
NEB's technical guide list which enzyme are methlyatble.
However I don't believe EcoRI is affected by either Dcm or Dam or even CpG meltylation.
XL1 blue is Dam+ Dcm-
However I don't believe EcoRI is affected by either Dcm or Dam or even CpG meltylation.
XL1 blue is Dam+ Dcm-
Thanks, perneseblue. I looked up the NEB guide and EcoRI is only susceptible to CpG if an overlapping site is present. This doesn't seem to be the case when looking at my primer sequences adjacent to the cloning site of pGEM. Any other ideas as to why EcoRI activity could be impaired?