3 replies to this topic

[thc]

Hi, I'm trying to transform e-coli cells, and it is not working.  I get only a few colonies, which is not enough to do colony PCR.  

I am using the PGEM T easy vector, and I am not even getting many negative colonies.  I followed the protocol my instructor gave me, although it wasn't very detailed.  I did some googling and realized I did a few things not quite right, but nothing that I feel should make me get so little results.  1) I defrosted the cells a little bit faster by warming them with my fingers, although I kept it on ice most of the time.  2) I centrifuged at 7600rpm 3 min, took off a bit of supernatent to increase concentration.  When I resuspended the pellet before plating, I probably caused some shearing with the pipet tip I used to resuspend.  (What is the best way to resuspend the pellet?)  

Another thing I can think of that might have gone wrong: when I plated the cells, I used an glass loop using 100% isoprop. to innoculate.  Perhaps I did not burn off all the isoprop., which killed the cells?  (How long should I keep it over the flame?).  

I trying to make sure I do it properly when I do this over again.

Thank you,
thc

Edited by thc, 08 May 2009 - 05:21 PM.

[kajmak]

Defrost the cells (in hands)
Add 50 µL of cell to ligation reaction
On ice for 30 min (15 works just as fine)
42 C for 60 sec
Ice for 2-5 min
If electroporating, use 1.8 kV and follow from here
Add 0.5 mL of LB broth (or SOC, I’m just lazy to make SOC)
37 C for 1 hr (45 min works)
Pellet at 14 000 rpm for 30 sec (using pGem T easy you don’t even have to concentrate them, sometimes colony separation is terrible if cell are pelleted here)
Resuspend them using tip, be gentle
Plate, use 100 % EtOH, cool spreader for 10-15 sec, spread cells (don’t overdo it or you’ll kill them) and let them be taken in by the media

[thc]

kajmak, on May 9 2009, 03:12 AM, said:

Defrost the cells (in hands)
Add 50 µL of cell to ligation reaction
On ice for 30 min (15 works just as fine)
42 C for 60 sec
Ice for 2-5 min
If electroporating, use 1.8 kV and follow from here
Add 0.5 mL of LB broth (or SOC, I’m just lazy to make SOC)
37 C for 1 hr (45 min works)
Pellet at 14 000 rpm for 30 sec (using pGem T easy you don’t even have to concentrate them, sometimes colony separation is terrible if cell are pelleted here)
Resuspend them using tip, be gentle
Plate, use 100 % EtOH, cool spreader for 10-15 sec, spread cells (don’t overdo it or you’ll kill them) and let them be taken in by the media

I don't think the spreader was too hot, and even if it were, it shouldn't kill all the cells.  

I understand the general protocol.  I'm trying to figure out what went wrong when I did it.  For some reason, a lot of cells died or the plasmid didn't ligate or something.

[kajmak]

Maybe competent cells are too old.
Try to do transformation with a plasmid as a control.  Just anything you have.  
Sometimes T overhangs on pGem T Easy fall off, due to often thawing-freezing.  One other thing, if you are not using Taq you need to add dATPs on PCR product.