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Sequencing of PCR product


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4 replies to this topic

#1 seed

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Posted 08 March 2012 - 01:20 AM

My PCR products are around 200-400bp. I would like to to gel extraction using freeze-squeeze method. Since that I got a bright and clear bands of my PCR products, I would like to do gel extraction, quantify my samples using nanodrop, and purify my samples before send for sequencing. I dont want to do any cloning because I jsut want to know wheter my PCR products are the right products. I'm going to send my samples to sequencing to Macrogen. I've read about sample preparation but I am still not really understand how to prepare my sample for sequencing. The company ask about the primer, should I send to them the primer of my specific gene of an universal primer (such as M13)...

#2 leelee

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Posted 08 March 2012 - 07:47 AM

Send them some of the primer you used to do the pcr, i also use macrogen and this is what I do.
Also, I'm pretty sure they do all of the purifying for you so you can skip the purification :)
(I've always just sent my per product untouched, just checked on a gel for size and approx concentration)

#3 bob1

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Posted 08 March 2012 - 11:54 AM

The universal primers are ones found on plasmids, as you havn't cloned your gene, it is unlikely that you will have this sequence at the start or end of your PCR product... use your gene specific primers. Note also that to sequence in each direction, you only add one primer to each tube - if you add both the competing reactions will give you an usable signal.

I would purify the PCR using a PCR clean up kit or ethanol precipitation if you have single band products.

Edited by bob1, 08 March 2012 - 11:55 AM.


#4 phage434

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Posted 08 March 2012 - 04:51 PM

Bob1 really meant "if you add both the competing reactions will give an an UNusable signal."

#5 bob1

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Posted 10 March 2012 - 12:59 PM

Bob1 really meant "if you add both the competing reactions will give an an UNusable signal."

Oops, yeah, me and my unreliable typing...




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