Dear All,
Im planning to use qiagen long range PCR and qiagen seqtarget system to amplify my gene of interest which is 40 kb in size.
The seqtarget provided 4 primers that covers the entire gene of my interest. And i manage to optimize the pcr for these 4 amplicons
But my problem are:
1. the pcr prod showed no bands if i use master mix
2. i get distinctive band if i add each reagents (dntp, q solution, taq, dH2O, primer) one by one (which means that i dont do master mix)
3. But wen i do many samples at a time , i tend to get faint bands.
I desperately need help in solving this issue.
Besides i need to pool these 4 amplicons into one before proceeding with Nextera XT kit for Miseq. So if u pool these faint pcr prod will it affect the pcr quality?
Im planning to use qiagen long range PCR and qiagen seqtarget system to amplify my gene of interest which is 40 kb in size.
The seqtarget provided 4 primers that covers the entire gene of my interest. And i manage to optimize the pcr for these 4 amplicons
But my problem are:
1. the pcr prod showed no bands if i use master mix
2. i get distinctive band if i add each reagents (dntp, q solution, taq, dH2O, primer) one by one (which means that i dont do master mix)
3. But wen i do many samples at a time , i tend to get faint bands.
I desperately need help in solving this issue.
Besides i need to pool these 4 amplicons into one before proceeding with Nextera XT kit for Miseq. So if u pool these faint pcr prod will it affect the pcr quality?