If you have a single bright band on your agarose gel, then you can clean and sequence from the PCR product. IF you have more than one band, then you can cut out and gel purify the band you want and sequence from that material. The only time you MUST clone before sequencing is when you think (or know) that you have a mixed sample of PCR products.

You can always clone if you want to have an archived sample of your PCR product, but it depends on what you will need for your project.

Good luck!

Dear Sadia Kanwal

You can do "colony PCR" when the bacterial colonies are screened directly by PCR (without DNA extraction). Small quantity of cells are transffered into a PCR mix (by sterile pipette tip).

You need a cleaned PCR product before Sanger sequencing to gain readable sequence.

Regards,

Marcela

If you have a single bright band on your agarose gel, then you can clean and sequence from the PCR product. IF you have more than one band, then you can cut out and gel purify the band you want and sequence from that material. The only time you MUST clone before sequencing is when you think (or know) that you have a mixed sample of PCR products.

You can always clone if you want to have an archived sample of your PCR product, but it depends on what you will need for your project.

Good luck!

Dear Sadia Kanwal,

I agree with Katie A Clark and Sanger method being the traditional method need the isolate and cloned. As it need the pure and cleaned sample for good sequence read. Kindly refer the following link

http://www.springerlink.com/index/K56910Q412RP0610.pdf

Dear Marcela ,

Thank you so much for your suggestion. I'm using DGGE techinque for bacterial profiling. The prominent bands are excised from the gel, followed by reamplification and then purification. the purified product is used for sequencing.

Regards

Dear Katie,

Wonderful suggestion. many thanks :)

Dear Daljeet ,

Thanks for sharing link .

Regards

@Amit ,Agreed .thanks