Purified PCR product - (Feb/28/2008 )
Greetings people,
I purified my PCR product using the PCR Clean up kit from Vivantis. When I took the OD reading and did the calc, its 305ng/ul. That's quite high. Fine. To send for sequencing, I only need about 20ng. Yes, I can dilute it but is it possible I get such high conc from the kit as I elute with 100uL TE. Ran gel on the purified product and its pure with the target I want and the band is nice.
Thanks all.
-jchchye-
Did you run a molecular weight marker of known concentration together with your PCR sample? If you did, then you can also compare your band intensity with the marker and estimate your sample's concentration. But, if your PCR worked nicely, it's very likely that the OD reading was right anyway.
-f2dU-
QUOTE (jchchye @ Feb 28 2008, 11:22 PM)
Greetings people,
I purified my PCR product using the PCR Clean up kit from Vivantis. When I took the OD reading and did the calc, its 305ng/ul. That's quite high. Fine. To send for sequencing, I only need about 20ng. Yes, I can dilute it but is it possible I get such high conc from the kit as I elute with 100uL TE. Ran gel on the purified product and its pure with the target I want and the band is nice.
Thanks all.
I purified my PCR product using the PCR Clean up kit from Vivantis. When I took the OD reading and did the calc, its 305ng/ul. That's quite high. Fine. To send for sequencing, I only need about 20ng. Yes, I can dilute it but is it possible I get such high conc from the kit as I elute with 100uL TE. Ran gel on the purified product and its pure with the target I want and the band is nice.
Thanks all.
If you have 305ng/ul and you have 100ul of it, then its like you have a total 30ug of PCR product. Its too much. Dilute the samples and also try to have add a ladder which can help you estimate the concentration of the PCR product.
-scolix-