4 replies to this topic

[gyma]

hello everyone. I have been having some trouble in amplifying a plasmid, which contains a full proviral genome of a retrovirus. Due to the presence of LTR and other unknown reason, I cannot get the correct plasmid. After transformation and miniprep, I digested the extracted plasmids and compared them with positive control, none of which has the correct pattern of digestion. I have tried two ecoli strains, DH5a and stbl3 and also tried different incubation temperatures such as 30, 33, 37c. Anyone has some advice? thank you.

[Christos Karamitros Oyama]

did you check whether the stains you used are Rec-? Meaning that , the gene encoding for recombinase has been deleted? If not, then most likely the cells recombine your plasmid and you get totally different sequence at the end.

This is the only logical I can think right now.

I hope that helps.


Best,
Chris

[ascacioc]

what origin of replication do you have on the plasmid? Maybe you need an additional factor such as pir for amplification of R6K origin.

And BTW (the genotype of stbl3 from http://openwetware.o...8Invitrogen.29:
STBL3 (Invitrogen)

F- glnV44 recA13 mcrB mrr hsdS20(rB-, mB-) ara-14 galK2 lacY1 proA2 rpsL20 xyl-5 leu mtl-1

• Streptomycin resistant
  
• endA+, use care in preparing DNA from this strain

That might explain why this strain does not work for amplifying plasmids:)

Andreea

[gyma]

Christos Karamitros Oyama, on 12 August 2012 - 05:44 PM, said:

did you check whether the stains you used are Rec-? Meaning that , the gene encoding for recombinase has been deleted? If not, then most likely the cells recombine your plasmid and you get totally different sequence at the end.

This is the only logical I can think right now.

I hope that helps.


Best,
Chris

DH5a is RecA1 while Stbl3 is RecA13, regarding this point, it seems DH5a is a little better. I did get "almost correct" clones from DH5a. But my labmate's success came from Stbl3 and thats why I switched to this strain. Maybe I need to pick more clones?

[gyma]

ascacioc, on 13 August 2012 - 04:55 AM, said:

what origin of replication do you have on the plasmid? Maybe you need an additional factor such as pir for amplification of R6K origin.

And BTW (the genotype of stbl3 from http://openwetware.o...8Invitrogen.29:
STBL3 (Invitrogen)

F- glnV44 recA13 mcrB mrr hsdS20(rB-, mB-) ara-14 galK2 lacY1 proA2 rpsL20 xyl-5 leu mtl-1

• Streptomycin resistant
  
• endA+, use care in preparing DNA from this strain

That might explain why this strain does not work for amplifying plasmids:)

Andreea

thank you very much for this information.