Long-range PCR

charvi · 12-04-2017, 08:36 PM

Hi everybody,
I try to amplify two long-range product about 11000bp each (one amplify exon 1-7 and one amplify exon 19-27), %GC about 62. All criteria for primer design is suitable except primer F for product one located in an region of Loc105371566 gene and primer R for product two is near DNA/TcMar-Tiger. I don't know whether these sequence affect my primer activity. Do anyone have experience about amplify product like this?

nucacidhunter · 12-05-2017, 12:54 AM

I do not know about specifics of your target regions. To be more successful you can try different primer sets around your region of interest and also consider a nested or semi-nested approach.

charvi · 12-06-2017, 12:56 AM

Thank nucacidhunter,
I'm considering semi-nested approach. However, my product is long. I'm worry it will take time to amplify my target. Normally, with product have length like that it takes about 5.5 hours. So If I apply semi-nested, What product length I should choose for outer primer to save time?

nucacidhunter · 12-06-2017, 02:32 AM

You need to walk out of region as far as you can get a good priming site and it does not have to be long.

finswimmer · 12-06-2017, 09:06 PM

Hello,

can you please explain a little bit more, why you think that this region is difficult to amplify? 11kb and a gc conten of 62% doesn't sound quite difficult to me.

fin swimmer

charvi · 12-08-2017, 12:53 AM

Dear finswimmer,
As I previously said in my question, my region is near LOC gene and repeat DNA/TcMar-Tiger. I don't know whether these sequence affect my results. I already had experience with amplifying so many long product more than 11kb and I had good results except these two products. Also, my primer is far away the splice site. So actually I don't know what happen except that clue.
I'm working with human genomic DNA

charvi · 12-08-2017, 12:59 AM

The one issue that I concern when apply semi-nested is time. I don't worry much about the product length as long as I can amplify my product with short time (less than 8hours).

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12-04-2017, 08:36 PM	#1
charvi Junior Member Location: Viet Nam Join Date: Dec 2017 Posts: 4	Long-range PCR Hi everybody, I try to amplify two long-range product about 11000bp each (one amplify exon 1-7 and one amplify exon 19-27), %GC about 62. All criteria for primer design is suitable except primer F for product one located in an region of Loc105371566 gene and primer R for product two is near DNA/TcMar-Tiger. I don't know whether these sequence affect my primer activity. Do anyone have experience about amplify product like this?

12-08-2017, 12:53 AM	#6
charvi Junior Member Location: Viet Nam Join Date: Dec 2017 Posts: 4	Dear finswimmer, As I previously said in my question, my region is near LOC gene and repeat DNA/TcMar-Tiger. I don't know whether these sequence affect my results. I already had experience with amplifying so many long product more than 11kb and I had good results except these two products. Also, my primer is far away the splice site. So actually I don't know what happen except that clue. I'm working with human genomic DNA Last edited by charvi; 12-08-2017 at 01:01 AM.

12-05-2017, 12:54 AM	#2
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	I do not know about specifics of your target regions. To be more successful you can try different primer sets around your region of interest and also consider a nested or semi-nested approach.

12-06-2017, 12:56 AM	#3
charvi Junior Member Location: Viet Nam Join Date: Dec 2017 Posts: 4	Thank nucacidhunter, I'm considering semi-nested approach. However, my product is long. I'm worry it will take time to amplify my target. Normally, with product have length like that it takes about 5.5 hours. So If I apply semi-nested, What product length I should choose for outer primer to save time?

12-06-2017, 02:32 AM	#4
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	You need to walk out of region as far as you can get a good priming site and it does not have to be long.

12-06-2017, 09:06 PM	#5
finswimmer Member Location: Europe Join Date: Oct 2016 Posts: 60	Hello, can you please explain a little bit more, why you think that this region is difficult to amplify? 11kb and a gc conten of 62% doesn't sound quite difficult to me. fin swimmer

12-08-2017, 12:59 AM	#7
charvi Junior Member Location: Viet Nam Join Date: Dec 2017 Posts: 4	The one issue that I concern when apply semi-nested is time. I don't worry much about the product length as long as I can amplify my product with short time (less than 8hours).