PCR Test and Gel for Ligation Test

Date Performed: August 3rd
Performed on Plates B, C, D, E

Step 11: Ligation Test
Pipette the ligation test sample(s) into new PCR strip tubes (40ul)
Perform a 1.5X bead cleanup for each test sample.
Briefly:
Add 60ul room temp KAPA Pure Beads to each sample and pipette mix 10X
Place on orbital shaker 15 min
Place on magnet rack and wait for solution to go clear
Remove clear solution without disturbing beads
Add 200ul fresh 80% ethanol to tubes
Remove solution from tubes
Add 200ul fresh 80% ethanol to tubes
Remove solution from tubes
Remove extra liquid with a p20 pipette
Wait ~2 minutes for excess ethanol to evaporate
Resuspend beads in 20ul nuclease free water
Place on orbital shaker for 5 minutes
Place on magnet rack and wait for solution to go clear
Remove clear solution from tubes into new strip tubes
For each ligation test tube, there will be 2 PCR reactions, a 12 cycle and a 30 cycle PCR
Make a PCR master mix on ice:
Thaw ready mix and primers on ice.
Vortex and spin down the ready mix and primers before use. Remember each ligation test gets 2 PCRs, so for 1 test samples the “n” number is 2.1 (error), for 2 test samples the “n” number is 4.2, etc. Use any primer set, 50X and 70X pair
10ul KAPA HiFi HotStart Ready Mix * “n”
4ul nuclease free water * “n”
0.5ul 50X primer * “n”
0.5ul 70X primer * “n”
Make one set of strip tubes for the 12 cycle PCR. Make a second set of strip tubes for 30 cycle PCR
Into each tube in both strip tube sets that is going to be used (depends on how many test samples you have) add 15ul of the PCR master mix
Add 5ul from the cleaned test sample(s) into both a tube in th 12 cycle set and the 30 cycle set
Vortex and spin down the tubes meant for the PCR
Use two thermocyclers, set the 12 cycle tubes in the thermocycler programed for the 12 cycle RAD LIG TEST program (in JONP Login). Set the 30 cycle tubes in the thermocycler programed for the 30 cycle RAD LIG TEST program
While that is running, make a small 1% gel to set (gel protocol)
After the program is done, run 10ul from each PCR reaction (use 2ul dye for 10ul sample) on the gel with a 1kb Plus DNA ladder for 1 hr at 80V
You should see a smear on the gel for all of the wells, the 30 cycle samples will be brighter, and might have brighter/more amplification at the smaller fragments (because smaller fragments amplify better in PCRs, which is why we only do 12 cycles in the real prep). The smear shows that the fragments got the adapters so they could be effectively primed and amplified to a level that you can see on the gel. If there is no smear for samples on the gel, it means the ligation did not work and you should go back to step 9 to do the ligation again.