I am using the Qiagen UltraRun Longe Range PCR kit to genotype zybrafish with expected bands of ~1800 and ~8000bp. I am using the PCR 2 step protocol provided with the Qiagen kit and see the ~1800 bp band but not the other. I've increased the annealing/extension time to 8 minutes (protocol states 30s/kb) from 4 mins but still don't see the 8000bp band. Any suggestions would be greatly appreciated.
Other information:
1% agarose gel used
No restriction enzyme used
PCR settings as follows
Initial deactivation: 93C x 3 min
2-step cycling (x30 cycles): 93C x 30 sec
65C x 8 min
Final Extension: 72C x 10min
Hello
To amplify an 8kb fragment, apart from having a good enzyme, having a DNA with high integrity is critical. You got your 1800bp fragment means annealing temperature is OK. You may try the 3-step PCR recommended by kit also.
Good luck
I would also say you're not amplifying anything with the current protocol you're using. I agree with Ali, either use a different protocol or try to get better quality gDNA. Good luck!
How long are your primers for your long band?
Did they have some restriction enzyme sites at the ends?
I also tried to PCR 10kbps, and finally got them.
The reason why PCR does not work is that your primers are not appropriate for making long PCR bands. So the first way is to change your primers for getting your long band.
Second, you should screen your several polymerase candidates for the best one for long PCR products.
I did try the 3 step PCR protocol and results were worse. Adjusted annealing temp but that did not change the outcome. The primers are 19 and 20 nucleotides.
Primer selection is limited with only 3 real options. I trialed the first set of primers (which did not work) and am using the second set now (I don't think the third set will work so probably won't bother with it). There is a large insertion (~6000 nucleotides) that contains a lot of repeating nucleotides.
This is the first time I've attempted long range PCR and haven't trialed any other kits. Sang-Min Paik is there one you've had better luck with?
Katie Pauly I used 'iNtRON, Maxime™ PCR PreMix (i-MAXⅡ)' about 5 years ago.
https://intronbio.com:6001/intronbioen/product/product_view.php?PRDT_ID=66&page=2&Scate1=2&Scate2=2&Scate3=1&Scate4=-263-264-265-266-&Scate5=&Scate6=&Sword=
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