Saturday 11/21/15
Added 8 uL of nuclease free water to Libraries 2, 3, and 4. Put in 4degC.
Sunday 11/22/15
Set up a PCR of wells H1-B4 of Library 1 (did not have enough Taq to do any more so ordered more Q5 High-Fidelity Taq). As the 100 uL PCR reactions recommended in the Meyer 2bRAD protocol always overfill my gel wells, I rescaled the recipe to be 77.75 uL reactions (50 uL ligation product + 3.75uL each barcode + 20.25 uL master mix).
First, made new working stocks of primers:
- Made 10 uM stock of Lib 1 and Lib 2
- 5 uL stock + 45 uL NFW
- Made 1 uM stock of BC11, BC12, HT2, HT3, HT4, HT5, HT6, HT7, HT8
- 1 sock + 99 NFW
|
1x
|
32
|
|
|
10 mM (each) dNTPS
|
2: 1.5
|
48
|
|
10 uM ILL-Lib1
|
2: 1.5
|
48
|
|
10 uM ILL-Lib2
|
2: 1.5
|
48
|
|
5X Q5 buffer
|
20: 15
|
480
|
|
Q5 Taq polymerase
|
1: .75
|
24
|
|
20.25
|
648
|
Added 3.75 uL of each barcode (this took a lot of time. I marked on the datasheet as I went to make sure I added barcodes to each well.)
Made a large gel with 24 wells and a regular gel with 8 wells. The top part of the large well broke so was only able to run 22 of the 31 PCRs (H1-C3). I put the gel slices in 1.5mL tubes with 40 uL of NFW and left overnight in the fridge.
Did the AlfI digestion of libraries 2, 3, and 4.
|
1x
|
230x
|
|
|
10x buffer R
|
1.2 uL
|
276
|
|
150 uM SAM
|
.8
|
184
|
|
AlfI (2 U/uL)
|
.5
|
115
|
|
water
|
1.5
|
345
|
|
4
|
920
|
Thermocycler at 37degC for 2 hours and 65degC for 15 minutes. Put in fridge afterwards.
Getting My Genes Wet 