that is a good strong clean product so I would expect it to sequence perfectly if it is the right size. The small amount of smearing is just caused by slight overloading of the sample.Go ahead and sequence

I think you can perform gel purification to get clean product.

that is a good strong clean product so I would expect it to sequence perfectly if it is the right size. The small amount of smearing is just caused by slight overloading of the sample.Go ahead and sequence

Dear Dr Aref,

I think that the amplicon are good to be sent for sequencing. However, if you are concerned about the smear, then I suggest you to slice the bands from the gel and purify using a good agarose gel purification kit before sending.

best wishes

SD

Thanks dear colleagues.

YES! OF COURSE QUANTIFIED AND WITHOUT PRIMERS IF IT IS A PCR PRODUCT

Thanks Dr. Bonito.

Just wanted to add, but have discovered that two people (Datta and Mebrahtu) have suggested my thoughts. Take the pains of performing gel purification again.

In diagnostics we routinley sequence PCR products like this. Your gel shows a clean PCR product! Do not worry about the little smear. I take it that you will use different primers for sequencing than you have used in your PCR. In the case that the smear would be a PCR by-product, the sequencing primers will select for the desired DNA fragment. No need for gel purifications!

good luck!

Thanks Prof. Berg and Dr. Ahmed. I'm going to send them for sequencing without gel purification. I'll try to share the results as soon as possible.

Try to purify the band from the gel via using gel purification kit, then send it for sequencing,

Good luck.

This is good amplification. If you sequence directly, you might have some noise because PCR reaction contains excess amount of primers and dNTPs. If you gel purify, yield might drop significantly. I would recommend to use one of the PCR purification column kit (generally less than 100 bp flow through) or use ExoSap-It (Exonuclease I) with pcr product and send it directly to sequencing. I've done both, both worked.

Hi,

To get neat and clean peaks without noise, you should purify it from gel.

Thank you.

I agree with jorg berg sir suggestion. No need of gel purificatio. Go ahead for sequencing.

good luck

Hi

this PCR product is so much good for sequencing after purification by gel extraction kit such as vivantis

Hello. I think it is an excellent PCR product for sequencing, go ahead

Hi

I suggest you to go ahead with direct sequencing. Its a real good PCR amplification with slight background. Don't worry about background, as Paul said, its because of overloading of the sample. Further adding to this, as Jorg said you can use an internal primer for sequencing reaction so that even there are non specific amplification you can avoid that and amplify your specific band in sequencing PCR.

Regarding the excess dNTPs and primers, after the sequencing reaction for Sanger method, there are washing steps to clear off these.

I was working with COX1 gene for bar-coding experiments where i used direct PCR product for sequencing.

Good Luck

Thanks Dear colleagues for your useful suggestions.

I coincide with other colleagues. These bands are good enough for sequencing. Just go ahead and good luck!

Yah, you can

It will be good to continue for down steams applications. sequencing will but you on the track.

Thanks all Dear colleagues.

I received the sequencing result yesterday. I had sent them without purification and now the result is OK.

Many thanks for letting us known that you had success with the sequencing of your PCR product! Congratulations!

Cheers Jörg

Dear Professor Berg,

You're welcome.

Dear Aref Alikhani

It is a good strong clean product so you go a head and do sequencing. I think you will obtained qualified result. You do great job. Best regard