sequencing of Pcr product - (Aug/25/2001 )
Is it possible to do a sequencing of a PCR product without cloning it into a vector.
You can sequence it using your PCR primers.
Yes, it is possible. But you will not get the full length of the product. Probably around 70-80%. Use Quiagen's QuiaQuick to purify your product. Very easy. Also, you'll need to use a large comb sizeso you can load around 30-40 ul of PCR product.
you can't sequence the PCR product directly due to the fact that the PCR sample contains among other thing BOTH your primers. So, you have to purify the fragment first. you can for example run it on a gel, excise the band, and pruify it.
Sure, we do it all the time. Isolate the PCR product from an agarose gel and clean it up with a kit (we use the Qiagen Qiaex II kit). You can use the same primers you used to amplify the PCR product with, and we use the USB Terminator Cycle Sequencing Kit. Good Luck!