6 replies to this topic

[Khudson0805]

Recently i was working with a vector which was methylated at the xbaI site. The vector was transformed into dam - cells in order to perform restriction digests with xba1 in the future.  I digested
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.

[Khudson0805]

I assumed it didn't work because the xbaI site was remethylated becaused I had used dh5a cells to transform my ligation. But colleagues of mine were advising that once DNA has transformed into Dam- cells it cannot be methylated if it is retransformed into other cell lines

[Curtis]

From NEB site:

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.

[leelee]

DH5a are dam- so that isn't your problem.

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.

[Khudson0805]

Our Z comp DH5a cells are dam +.

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

[leelee]

Huh...I wonder if I might be getting confused, I was sure my DH5a are dam-...unless I'm mistakenly thinking of another type of methlation...wish I had my lab book at home so I could get this straight in my head! Whoops.


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

[Khudson0805]

thanks so much! just ended up amplifying new restriction sites I am not sure the vector even cut properly because when I sequenced what I thought was a ligation it turned up to be just the vector and no insert! The MCS was still intact