PCR Gradient Protocol in T100™ Thermal Cycler
You will need to make up 8 identical PCR tubes for every DNA sample of interest. The T100™ Thermal Cycler machines in the MEE lab do gradients only over 8 temperatures.
For a 10 ul reaction, you will need an identical tube of same reagents with the same volume in every row from 1 to 8. For the range, Row 1 is the highest/hottest temperature (Red) and Row 8 is the lowest/coldest temperature (Blue). You decide the highest and lowest temperatures of the range and the machine will automatically fill in the 6 temperatures within the range.
Making 8 Identical 10 uL Reaction Tubes with Master Mix
Reason for Making Master Mix Instead of manually pipetting each reagent into tubes to make up many PCR reactions, it is more convenient to make a master mix and divide it out. When mixed well, master mixes increase uniformity across tubes and decrease the chances of human error because of less pipetting and working with larger volumes.
In order to make up the 8 identical tubes, you will need to make a master mix containing all the reagents and then divide them into the 8 PCR tubes.
NOTE you will make a master mix for 9 tubes (1 extra tube added) to account for human error and to avoid ending up with a tube that does not have enough. Furthermore, once all 8 tubes are full, you can perform and extra tube test to see if the remaining amount is exactly 10 uL left over.
1) Calculate the volume of each reagent for 9 times (x9) the amount of reagents needed for a 10 uL reaction. 2) Get a 0.2 mL microcentrifuge tube and add in reagents for master mix.
a) Be sure to vortex the reagents before adding them to the master mix tube.
3) Once the master mix tube is made up, vortex it/ 4)Centrifuge the master mix tube. 5) Get an 8-strip of 0.2 mL PCR tubes and a coverslip.
6) Add 10 uL of the master mix into each tube.
7) When all the 8 tubes are filled, do an EXTRA TUBE TEST by pipetting up the final volume in the master mix tube. If the remaining volume...
a) seems like it is roughly 10 ul, your master mix is probably fine. b) falls a little short of 10 ul, maybe the volume distribution in the 8 tubes was not equal. You can probably proceed. c) is close to 0 ul, you might have not added the proper volume for all reagents. i) You might have to redo the master mix to avoid an unsuccessful PCR ii) If you really trust that you put all reagents, then make a note and proceed.
8) VERY IMPORTANT: Label the coverslip of the 8-strip to distinguish one end from the other (e.g Hot end and Cold end) KEEP IT THE SAME ORIENTATION EVERY TIME YOU PUT IT ON THE TUBES 9) Vortex and centrifuge the 8-strip of tubes Input the appropriate settings for the reaction in the PCR machine 10) This will include adding the highest and lowest temp of the range 11) Take note of the temperature range