Hi Dear Researcher,
I have a problem with transformation. Actually I do not know where is the problem. I am trying maybe 80th times but I am not finding true bands view .
I want to tell whole process.
I used head shock method. 100 ul E.Coli DH5α and 2 ul plasmid DNA (pCDF1-JAK2-GFP 10241 bp)
30 min. ice--> 1 min. 42 °C --> 2 min. ice --> 900 ul SOC media --> 1 hour 225 rpm 37 °C shaker -->Spreading to Agar Plate (fresh ampicilin also I added extra ampicilin)
12-14 hours inc.
I am getting a lot of colony.
Seeding to 5 ml LB + amp --> 8 hours 225 rpm 37 °C shaker --> Miniprep (used QIAgen minikit)
I am gettin good amount of DNA
There is no problem so for.
After enzyme digestion ( I tried 3-4 different enzyme 7-8 different configuration) I am facing incorrect bands and result.
I really need your suggestions. Please help me about this problem
*I checked LB, LB agar, ampicilin amount, temperatures, E. Coli efficiency, enzyme efficiency, used uncut control and positive control.
neoplaziMember Since 11 Feb 2013
Offline Last Active Feb 21 2013 07:07 AM
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My research interests
Myeloproliferative Neoplasms-Stem Cell
25 Feb 2013 - 12:55