Problem on PCR from cDNA - (Nov/28/2006 )
Hi everyone!
I used Pfx (invitrogen) to amplify a 1.5kb fragment from C.elegans cDNA with the Mg concentration at 2.5mM, 3mM, and 3.5mM. For unknown reason, my PCR product on the next day was very viscous!!! Has anybody here ever met this before? My PCR condition is 94C 3min, (94C 30sec, 55C 30sec, 68C 1min50sec for 30 cycles) 68C 10min, 4C
Thanks a lot!
-flametree-
may be u need to add more dH2O......since it's a 2 step-PCR (reverse transcription+amplification)..
-strawberry-
QUOTE (flametree @ Nov 28 2006, 12:10 PM)
Hi everyone!
I used Pfx (invitrogen) to amplify a 1.5kb fragment from C.elegans cDNA with the Mg concentration at 2.5mM, 3mM, and 3.5mM. For unknown reason, my PCR product on the next day was very viscous!!! Has anybody here ever met this before? My PCR condition is 94C 3min, (94C 30sec, 55C 30sec, 68C 1min50sec for 30 cycles) 68C 10min, 4C
Thanks a lot!
I used Pfx (invitrogen) to amplify a 1.5kb fragment from C.elegans cDNA with the Mg concentration at 2.5mM, 3mM, and 3.5mM. For unknown reason, my PCR product on the next day was very viscous!!! Has anybody here ever met this before? My PCR condition is 94C 3min, (94C 30sec, 55C 30sec, 68C 1min50sec for 30 cycles) 68C 10min, 4C
Thanks a lot!
Did you check that you actually obtained an amplicon? if so i wouldn´t worry too much but more details on what your enyzme mix is . I´m thinking that as already mentioned in the post above you have not added sufficient water to dilute down the glycerol which is used to store enyzmes.
-Ritchy-
Here's my reactioin system: 3ul cDNA, 1.5ul 10mMdNTPs, 1.5ul 10uM Forword primer, 1.5ul 10uM Reverse primer, 5ul 10x buffer, 2-3ul 50mM MgSO4( I'm optimizing the Mg concentration), 0.4 ul Pfx, 36-35ul H2O. Total volume is 50 ul.
-flametree-