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Cloning


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#1 predoc

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Posted 14 June 2010 - 10:57 AM

I have a 10.5 kb vector in which I am introducing a mutation with PCR primers. I designed primers such that I have the required mutation and also a sticky end restriction enzyme site, AvrII.
I did the PCR, I ran 3 ul of the sample and saw the 10.5 kb band. I took 10ul of the PCR mix, put AvrII and DpnI. I gel extracted it.
Then I ligated o/n with T4 DNA ligase at 4C. I ran 5ul of this reaction on a gel. I still see a band of 10.5 kb.
Then I transformed it along with my original vector as positive control. I saw a gazillion colonies.
I grew 1 colony from my positive control and 8 of the experimental.
After mini-prep, I ran 5 ul of the reaction just to see if I had plasmid, but I saw nothing. I haven't gotten to checking the plasmid by restriction digestion becoz I see no plasmid on the gel. What might be going on?

Help!!!
:lol:

#2 ranvi

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Posted 21 June 2010 - 01:52 PM

may be the concentraion of the plasmid is too low to see on the gel?
try loading may be 20ul of the miniprepd plasmid

#3 Curtis

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Posted 23 June 2010 - 08:43 PM

it happens to me some times also, that's why I always pick more colonies and grow in different broth and extract separately. no worries.....my advice to you pick another colony and regrow in broth.

also, I have noticed that some times when I load my sample on the first well of the gel it looks so faint. so next time load in the middle wells.

#4 predoc

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Posted 28 June 2010 - 12:38 PM

Thanks guys!!

it happens to me some times also, that's why I always pick more colonies and grow in different broth and extract separately. no worries.....my advice to you pick another colony and regrow in broth.

also, I have noticed that some times when I load my sample on the first well of the gel it looks so faint. so next time load in the middle wells.






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