Dam-/+ e. coli strains
Posted 16 August 2012 - 05:16 PM
The vector was wirh xba and notI and it was used in a ligation rxn. I retransformed into dh5a cells. these cells are dam+ correct? this would mean that the Xba site would be remethylated? After i Miniprepped my ligation rxn I performed a digest to ensure that the insert was there. I used Xba and not and when I ran the gel jt just looked like vector.
Posted 16 August 2012 - 05:19 PM
Posted 16 August 2012 - 06:02 PM
Q1: Is XbaI affected by methylation?
A1: XbaI is blocked by overlapping dam methylation. If the recognition site is preceded by GA or followed by TC dam methylase GATC will overlap the XbaI site TˆCTAGA: blocked are ga TˆCTAGA and TˆCTAGAtc. To remove dam methylation use a dam deficient strain like GM2163 (E4105S).
The methylation must be removed. Maybe there is a problem during your digestion.
Posted 16 August 2012 - 06:35 PM
When you say it just looked like vector, do you mean it looked like uncut plasmid? Or did the NotI digest work so you saw only a linear band of vector size?
And your colleague is wrong.
When you transform your plasmid into a dam+ strain, and it starts to replicate your plasmid, the plasmid will be methylated (or not) according to the genotype of that strain. Regardless of if the plasmid you transformed in was methylated or not.
Posted 17 August 2012 - 04:36 AM
Correct it was just uncut plasmid- there was no obvious signs of my insert. The XbaI/NotI Digestion should have yielded a 800 bp fragment, but it was not there. Just vector. I am thinking because the XbaI site was methylated after retransforming the ligation into the dam + cells. I have been using the pLVX tight Puro vector and the TCTAGATC is present in the MCS
Posted 17 August 2012 - 07:01 AM
At any rate, the NotI should still have cut.
Have you tried doing just a single digest with NotI? If your insert is 800bp, you should be able to tell the successful transformants
Posted 22 August 2012 - 07:32 AM