subcloning

Misako Haraguchi haraguci at KHOSP2.KUFM.KAGOSHIMA-U.AC.JP
Wed Dec 4 23:24:34 EST 1996


I would like to ask some questions about subcloning.

I have not suceeded to do subcloning my gene into plasmid vectors for 4
monthes.   I tried to cut genomic DNA including my gene with SpeI and BglII
and ligate into BamHI and XbaI sites of PUC19.   At first, I extracted
about 100ng of fragment DNA (8kb) from agarose gel with QiaEXII or gene
clean spin kit.   I tried to ligate it into 100ng of PUC19 with ligation
high(TOYOBO).   Then, I tried to transform ligaton product into DH10B
electromax (Gibco BRL) by electropolation.   I obtained  about 300
colonies.   But the growing of these colonies was very slow, and very
small.   There were no positive clones.    

1. Do you have the information about efficient  DNA extraction methods from
agarose gel?

2. Do you have the information about efficient ligation kit?

3.  Do you have the information about host  E coli with high efficientcy?

4. How many colonies should I obtain after transformation? 1000 or 10000?

5. Do you have any check system whether you could ligate your gene into the
vectors?

6. Do you have any general recommendations about subcloning?


Thank you very much for your trouble.

Sincerely yours
Misako Haraguchi, Ph.D.
Departmemt of Cancer Chemotherapy ,
Institute for Cancer Research,
Faculty of Medicine, Kagoshima University ,
8-35-1 Sakuragaoka Kagoshima 890 JAPAN
tel: +81-992-75-5490
fax: +81-992-65-9687





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