Transformation problems
Joe Russert
Friday, 01 February 2008 21:36 UTC
I am having trouble with my transformations, I have been for the past month. In the past I have had successful transformation with little trouble.
Currently no colonies are growing in either control(ligated vector) or experimental(ligated vector and insert) transformations.
I transformed uncut plasmid last night to check my dh5alpha cells, and many colonies developed.
Does anyone have any suggestions?
Vector: pMES
Insert: mus Sphkinase 1 and 2
Ligase: Roche rAPid ligation kit
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Replies
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If you are getting colonies with uncut plasmid then your cells should be OK.. but sometime colonies can also be developed with non-competent cells..
You can check your ligation first.. like inactive buffers, enzyme or if digested vectors are not of compatible ends..
what end ligation is this?
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I am using 5’ XhoI sticky end, and 3’ EcoRI blunt end. I used my PCR to add the XhoI site, and cut the vector in a two stage single digest with Roche enzymes.
I always aliquot my ligation buffer, so I am confident that the ATP was not destroyed, but my old kit did just run out, so I will ligate with new buffers, and enzyme today.
Thank you very much for the input!
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Sorry, I actually used a 3’SmaI blunt end for this vector.
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I’ll check my digestion and ligation first by runnig the following controls..
For digestion:
1. untreated DNA-to demonstrate the integrity of starting material.
2.No enzyme control-can detect the changes may occur independent to the enzyme.
3. Independent enzyme activity control-control digest of DNA-to confirm the enzyme is active.
4. DNA substrate control-experimental digest-to compare the activity of enzymes under experimental conditions.Run on 0.8% gel
if digestion is fine.. then..
For Ligation, I’ll first analyse the linear(cut)vector DNA and ligated product on gel.. if ligation was successful then you will see different band patterns..
good luck..
Amit -
If you can purify your vector and insert by kits in your lab, maybe you can improve ligation. at the same time to make ligation time longer. have a good luck.
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yeah.. longer time will help..
yeah.. longer time will help..coz rate of ligation with sticky ends will be faster than the blent end ligation..
yeah.. longer time will help..coz rate of ligation with sticky ends will be faster than the blent end ligation..but overnight at 4 degree is fine..
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Thank you very much for the advice. My transformations are working now. I don’t know if my technique or my old kit was to blame, but good results are good results. Thanks again.
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