Cant get desired fragment from plasmid - confused!!!!!!!!!!! (Jun/24/2006 )

Hi there all,

Im doin biodiversity studies. Im using pDrive from qiagen for my ligation n cloning. I check my inserts using the Eco R1 enzymes. After that i pcr out the desired fragment which is about 973bp from the plasmid to be sent in for sequencing. this method has worked well till lately. Out of 38 positive tested plasmid only 8 shows the desired band after PCR.

I dont know y such a low count. Is it the ligation problem or my PCR problem? I never had any problem with PCR till lately. A labmate of mine suggested that i dilute the DNA before PCR although i have never diluted my DNA before n never had any problem. Isnt there a standrad method for PCR?

Pls help.Seriously confused.

PCR is one of those techniques where less is sometimes better. Try the sample dilution thing (although why it should start going strange now is a bit of a mystery...)
I presume you haven't done anything different in your preparation.
Try changing to fresh reagents, including water. Check to see if the water purification cartridge has been changed recently (I once lost about a month's work time because the cartridge was dirty, and the water quality went downhill- from great PCR to zip in a week!). Use a fresh dilution of dNTPs.
You can always ask sopmeone else in the lab to do the expt for you once. Another pair of hands can sometimes work wonders...

[Hi thanks for your advice. Im thinking of diluting the samples first, then ask someones help.

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