11/21/15 and 11/22/15

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.

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