5 replies to this topic

[beyonka8@gmail.com]

I cloned 2.1kb fragment into 5.4kb plasmid, I did transformation into BL21 and plated ligation reaction onto LB+Kanamycin 70. I got really good transformants. I made sure that they have insert using colony PCR, I also transferred colonies onto LB+Kanamycin 70 to make a master plate and incubated my master plate at 37*C But, my master plate the clone of insert (and others) doesn't seem to grow. Even after extended period of incubation. PLease guide
-pGenMutant.

[bob1]

It may be that they are false positives - if you picked the colonies off the plate and PCRed directly, you may well have picked up DNA from the transformation that was on the plate.

It could be that the plasmids are toxic to the bacteria.

[pDNA]

do they also fail to grow in liquide medium?

I would not use BL21(DE3) for cloning ...rather use a cloning strain that does not express your gene of interest and then subclone your plasmid into your expression strain once you have confirmed your insert.

Maybe your protein is toxic ...and they are able to grow once on solid medium ...but then get rid of the plasmid rapidly ...and the cells you use for inoculation are already dead or do no longer contain the plasmid.

What kind of gene you tried to clone and express?

Best,
p

[gyma]

are you sure you used the same kanamycin plate? just a simple transfer procedure, i dont think there are so many possible reasons. Maybe you used ampicilin plate.

[Christos Karamitros Oyama]

What primers did you use to run your colony PCR? if you used the specific of your gene, it could be the case that you amplified your gene which is present in the plate after the cloning step and remained unligated in your vector. The best, combination would be T7 FW and RV specific for your fragment or vice versa. I agree  with bob1.

Additionally, another step of confirmation would be to control digestion with your restriction enzymes you used for digestion. Did you try liquid culture?

Last, but not least, I would agree also with pDNA; do not use BL21DE3 for your cloning. In case that your protein is toxic you could use specific strains whose genes responsible for toxicity have been deleted. Strain like these is C41 or C43.

I hope that helps.


Best,

Chris

[ascacioc]

I agree with what people above mentioned related to cloning directly in BL21(DE3): it does not work as you would like it to work:) Unless you are using BL21-Gold(DE3) which is another story.  Better use DH5alpha or XL-1Blue as a cloning strain, recover the plasmid and transform it in BL21(DE3). Another thing: do you use SOC media for outgrowth? How long do you recover? SOC media is important since it contains glucose which inhibits the expression of your potentially toxic protein.

What is this LB-Kanamycin 70? Are you using 70 ug/mL kanamycin? The recommended concentration is 30-50 ug/mL final concentration. 70 would a tad too much.

Andreea