Successful Transformation->Getting Colongy--> Getting DNA after miniprepTransformastion
Posted 11 February 2013 - 04:52 AM
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.
Posted 11 February 2013 - 06:58 AM
Posted 11 February 2013 - 08:48 AM
So I am checking bands from it.
Posted 11 February 2013 - 10:35 AM
Posted 11 February 2013 - 10:22 PM
We took from different group that has been worked long time.
So there is no problem with construction of plasmid.
Posted 11 February 2013 - 11:30 PM
Posted 12 February 2013 - 05:47 AM
Posted 13 February 2013 - 06:22 AM
Thanks for your interest.
You are kind and helpful.
After your posts I digested main (stock) plasmid with other transformed colony miniprep results.
Here my gel image:
I was finding extra and incorrect band (end of hole) and I saw it in stock plasmid.
Do I have plasmid contamination?
I do not have sequence posibility for now.
Can you offer another suggestion?
Edited by neoplazi, 13 February 2013 - 06:58 AM.
Posted 13 February 2013 - 06:32 AM
Posted 15 February 2013 - 07:26 AM
Good bless me
Edited by neoplazi, 15 February 2013 - 09:14 AM.