Cloning purified PCR products eluted in Qiagen's EB - Concentrate a purified PCR product eluted with EB? (Jan/31/2009 )
kmkocot on Feb 2 2009, 02:45 PM said:
Thank you for the replies. I have a few PCR products that produced faint bands that I gel purified. I am using Promega's pGEM-T cloning kit so I want to use as much of my product for the ligation as I can.
hi,
TA cloning is easy. I think a visible band of DNA is sufficient for cloning. You don't need to concentrate it.
If you want to, a NaOAc/2-propanol precipitation can do the job.
-WHR-
tyrael on Feb 12 2009, 02:56 PM said:
i was using Vivantis GF-1 Nucleic Acid Extraction Kit to purify my DNA.....
however i was in a dilemma during the last step, i.e.: elution of DNA..
i was supposed to use the EB (elution buffer) to elute, however, the sequencing company that i would send to, request me to store my purified DNA in water.
so which one does matter ? as mentioned above, water might have uncontrolled pH, while EB has slightly alkaline pH...
any comments, pls ?
thank you.
however i was in a dilemma during the last step, i.e.: elution of DNA..
i was supposed to use the EB (elution buffer) to elute, however, the sequencing company that i would send to, request me to store my purified DNA in water.
so which one does matter ? as mentioned above, water might have uncontrolled pH, while EB has slightly alkaline pH...
any comments, pls ?
thank you.
I elute in TE, and dilute at least 20 times in water to sequence.
you could elute in tris buffer (EDTA will inhibit the PCR, unless it is enough diluted), and prepare the dilution for sequencing in water.
-little mouse-