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Help with transformation


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#1 thc

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Posted 08 May 2009 - 05:19 PM

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.


#2 kajmak

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Posted 09 May 2009 - 02:12 AM

Defrost the cells (in hands)
Add 50 ĶL 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, Iím 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 donít 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 (donít overdo it or youíll kill them) and let them be taken in by the media

#3 thc

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Posted 09 May 2009 - 11:27 AM

Defrost the cells (in hands)
Add 50 ĶL 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.

#4 kajmak

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Posted 09 May 2009 - 02:57 PM

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.




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