Index additions, cleaning, quantification

Date Performed: July 22nd

PCR and Index Addition USE FILTER TIPS FOR ALL PIPETTING
For each pool you are going to set up 6 PCR reactions. This minimizes possible PCR biases but also amplifies the libraries to a good amount. If you are amplifying more than one pool at a time (say 5), it can be easier to carry out all the reactions on a 96 well plate instead of individual strip tubes
Set up a plate or however many tubes you’ll need to have 6 wells/tubes per pool. Label A, B, C etc as these are very temporary
Thaw size-selected samples, ready mix, and 50X, 70X primers on ice. Vortex and spin down all before using
Each pool needs a master mix because each pool gets their own 50X-70X primer pair
Make a master mix for each pool (ex for 5 pools, you would make 5 mixes, each with their specific primer pair):
10µl of 2X KAPA HiFi Hot Start Ready Mix * 6.1 = 66
4µl water * 6.1 = 24.6
.5μl 20uM PCR primer 50X *6.1 = 3.3
.5μl 20uM PCR primer 70X *6.1 = 3.3
Vortex and spin down the master mixes
Pipette 15ul of each master mix into their planned 6 tubes or wells
Add 5ul of DNA from a size-selected pool to their respective wells, and repeat for the other pools
Cover the plate with a foil seal, vortex to mix, and spin down
Put in the Thermocycler for amplification, 12 cycle RAD PCR program under JONP login
Samples can be frozen afterwards, or continue to clean-up


Pool 1 –> 502/702
Pool 3 –> 505/705
Pool 4 –> 506/706
Pool 5 –> 507/707



1X Cleanup after PCR
Take out KAPA Pure Beads ~30 minutes before use and make fresh 80% ethanol
Combine the 6 reactions into a single 1.5 mL tube, for a total of 120μl
Add 1.5X of KAPA Pure Beads (180μl) to each pooled tube, mix by pipetting, and incubate on a shaker for 15 minutes
Put the tubes on the long magnet rack, wait until the liquid becomes clear, and remove as much of the clear liquid as you can
Add 1000μl 80% EtOH to each tube while keeping it on the magnet
Remove 1000μl of the EtOH
Add 1000μl 80% EtOH to each tube
Remove ALL EtOH carefully from each tube, use the p20 or p200 to remove any excess ethanol
Let samples sit for ~5 minutes, letting the ethanol evaporate, but the beads not dry out
Take tubes of the magnet stand and elute the DNA in 35μl 10mM Tris HCl and incubate shaking for 5 minutes
Place the tubes back in the magnet, and wait until the liquid goes clear. Remove all liquid and put into new strip tubes: this is your library!\

Quantify and Validate
UseBR dsDNA Qubit protocol. Run this twice, as in re-make the standards and new sample tubes

Since all failed I went back to Qubit sample cleaned pools pre-size selection (Step 12)

I should have included pool 2, but forgot. As I preformed all the previous steps the same I assume pool 2 also does not have enough DNA