I frequently experience that it does not work while preparing large volumes like 50 μl of PCR mixture, though the same ratios of chemicals operate when I make little amounts (15 μl) of PCR chemicals. What's the probable cause of this issue?
pcr machines are often set at an expected volume lower than 50ul. Smaller samples take less time to reach temperature so moving from 72 to 94 may only reach 90 before cooling to annealing begins so denaturation is not complete and less pcr product is generated. If you have a pcr machine that lets you define the reaction volume then this is less likely to happenas the machine allows longer to reach temperatures. It may also be that cooling to annealing temperature also does not reach the annealing temp so primer binding is poor
I have experienced of working large volume of 50 microliter in PCR and its works at this volume. You need to adjust the volume of each PCR component according to 50 microliter reaction volume.
There could be several reasons why a large volume of a polymerase chain reaction (PCR) mixture fails to operate effectively:
1. Insufficient Enzyme
2. Uneven Heating/Cooling
3. Suboptimal Primer Concentration
4. Inadequate Mixing
5. Inhibitors
6. Thermal Stability
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