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Ligation/Transformation Mess-up, are they going to survive?


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#1 KirithSoldier

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Posted 05 June 2013 - 02:11 PM

After putting together a ligated plasmid, I absent-mindedly put 250ul SOC into the ligation mix instead of into the cells. WHY I even had the SOC out yet to begin with I don't know, it was a rough day. My question is will the SOC keep the cells from uptaking the ligate? It was a quick transformation using Z-competent cells, and they're normally pretty forgiving... They normally need to be iced 20 minutes after ligation mix is added, then spread onto the plate with or without recovery.... I threw the cell/SOC/ligate onto ice 20 minutes as usual, but I'm worried the SOC will hinder the reaction. I guess I"ll find out tomorrow! But if anyone has any insight please comment :)

#2 bob1

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Posted 06 June 2013 - 02:58 AM

The SOC will dilute the ligation such that the transformation efficiency will probably be quite low. How are you doing the transformation - heat shock? electroporation? If heat shock, they may well be OK, just low efficiency. I would doubt that electroporation would work though.

#3 KirithSoldier

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Posted 06 June 2013 - 09:44 AM

well, it was a moot point because i plated them on the wrong antibiotic lol. what a day :/ but all is well and fixed now.

as far as the transformation method, we use chemically competent cells, so its super easy peasy. All you do is add up to 5% volume in ligate, ice 20 min, and plate. No recovery necessary, but I usually do it anyway.

Edited by KirithSoldier, 06 June 2013 - 09:45 AM.


#4 bob1

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Posted 06 June 2013 - 05:07 PM

Chemically competent cells are usually transformed using a heat shock (typically 42 deg for 1 min or 37 for 5 min), ice for 2 min and then allow expression of the antibiotic resistance for 1hour at 37 before plating. If you struggle for transformation at any point - you might want to think about the above...

#5 KirithSoldier

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Posted 06 June 2013 - 11:59 PM

I followed the protocol given to me by my PI about the particular cells he uses, and it seems to work for everyone else....but I will keep that in mind, thank you :)

#6 notjustalabtech

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Posted 09 June 2013 - 11:17 AM

Your transformation efficiency would probably be reduced, but you can just put all of your cells on the plate rather than just 100 ul of your final volume. Spin the tube down (after your 1 hr incubation and you are ready to plate) to reduce the volume, 8000xg for 3 min should do the trick. Decant gently, and use the remaining liquid to resuspend the pellet, then add all of it to your agar plate and allow to grow overnight.




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