Transformation troubles

mmustafa · Post by **mmustafa** » Tue Feb 26, 2008 9:05 pm

Transformation troubles

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Post by mmustafa » Tue Feb 26, 2008 9:05 pm

Hi All!!

I am struggling with trying to get my plasmid into competent cells. I am currently using the Invitrogen Gateway Directional TOPO kits and One Shot TOP 10 competent cells and have had no success with the transformation process. Any clues anyone?????

Cat · Post by **Cat** » Tue Feb 26, 2008 9:55 pm

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Post by Cat » Tue Feb 26, 2008 9:55 pm

How about +ve control???

mmustafa · Post by **mmustafa** » Tue Feb 26, 2008 9:58 pm

Re: Transformation troubles

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Post by mmustafa » Tue Feb 26, 2008 9:58 pm

I used the control plasmid provided with the competent cells. No colonies there either!!

Cat · Post by **Cat** » Tue Feb 26, 2008 10:15 pm

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Post by Cat » Tue Feb 26, 2008 10:15 pm

O.K., you have too many things that can go wrong - anything in the procedure to bad cells. The most common problems: 1. Heat shock: too long, wrong temp; 2. wrong antibiotic in selection media. If you are sure that you did not make mistakes in the procedure, then you can assume bad cells.

mmustafa · Post by **mmustafa** » Wed Feb 27, 2008 12:48 am

Re: Transformation troubles

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Post by mmustafa » Wed Feb 27, 2008 12:48 am

I have been heat shocking for 30 to 45 sec at 42C. That should be okay, right?

Cat · Post by **Cat** » Wed Feb 27, 2008 12:53 am

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Post by Cat » Wed Feb 27, 2008 12:53 am

That should be fine. Check the procedure step by step and see if you are doing anything different:

http://www.invitrogen.com/content/sfs/m ... 10_man.pdf

mmustafa · Post by **mmustafa** » Wed Feb 27, 2008 1:14 am

Re: Transformation troubles

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Post by mmustafa » Wed Feb 27, 2008 1:14 am

Nope! Nothing's different!!!!

Cat · Post by **Cat** » Wed Feb 27, 2008 2:59 am

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Post by Cat » Wed Feb 27, 2008 2:59 am

Then your problem is with competent cells.

mmustafa · Post by **mmustafa** » Wed Feb 27, 2008 3:32 pm

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Post by mmustafa » Wed Feb 27, 2008 3:32 pm

I got new stock of competent cells and performed transformation using a pUC19 control plasmid and got really tiny colonies. What does that mean??

Cat · Post by **Cat** » Wed Feb 27, 2008 4:46 pm

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Post by Cat » Wed Feb 27, 2008 4:46 pm

If there are many small colonies, then your transformation probably worked and size problem is likely due to too much antibiotic in the media. If there are only few small colonies, then they are likely to be escapees...

h2so4hurts · Post by **h2so4hurts** » Wed Feb 27, 2008 9:17 pm

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Post by h2so4hurts » Wed Feb 27, 2008 9:17 pm

Haha, or he didn't let the plates grow long enough...

Also, try adding 4uL of your PCR product into to TOPO reaction, especially if your PCR product is weak. Maybe try gel purifying your PCR product before the TOPO reaction.

mmustafa · Post by **mmustafa** » Thu Feb 28, 2008 3:15 pm

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Post by mmustafa » Thu Feb 28, 2008 3:15 pm

I got really nice strong single band (correct size) when I ran my PCR product on a gel. That's why I was confident that the transformation would go smoothly!!