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Hello!
I am having A LOT of trouble getting amplification from bar-coded primers using the Lib-L library set up. I have 9 biofilm extractions and attempting to create 9 tagged libraries for a 454 run. I am using 515F and 909R for my template specific primers. My biofilm sample amplify quite well using an annealing temperature of 56 using the STANDARD primers (i.e. no tags). However when I attempt the reaction using tagged forward primers 1-8 and the reverse pyrotaged 909R I have no amplification. The odd thing is that using forward primer number 9 I am able to achieve amplification on all of the samples.
Could there be something about the bar-codes used in relation to the different templates? I ordered the primers from invetrogen even though Roche suggests IDTdna. Not sure that maters...
Also, I used a 1:10 dillution of PCR product from the standard primer set as template for the tagged reactions 1-8 and was able to get some rough looking non-specificy-amplification along with a band in the right spot. However I would like to amplify directly from the template.
Any thoughts or suggestions would be greatly appreciated. I have been doing this kind of research for 10 years and have never come across a problem like this. Tested everything I could imagine to test/check/swap/recheck. Killing me.
Thanks
I am having A LOT of trouble getting amplification from bar-coded primers using the Lib-L library set up. I have 9 biofilm extractions and attempting to create 9 tagged libraries for a 454 run. I am using 515F and 909R for my template specific primers. My biofilm sample amplify quite well using an annealing temperature of 56 using the STANDARD primers (i.e. no tags). However when I attempt the reaction using tagged forward primers 1-8 and the reverse pyrotaged 909R I have no amplification. The odd thing is that using forward primer number 9 I am able to achieve amplification on all of the samples.
Could there be something about the bar-codes used in relation to the different templates? I ordered the primers from invetrogen even though Roche suggests IDTdna. Not sure that maters...
Also, I used a 1:10 dillution of PCR product from the standard primer set as template for the tagged reactions 1-8 and was able to get some rough looking non-specificy-amplification along with a band in the right spot. However I would like to amplify directly from the template.
Any thoughts or suggestions would be greatly appreciated. I have been doing this kind of research for 10 years and have never come across a problem like this. Tested everything I could imagine to test/check/swap/recheck. Killing me.
Thanks