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November 19 – Test and Final PCR Reactions (RADSeq)

This week we ran two PCR reactions. One was a test to see if it would work and the other was the final one for our DNA samples we have been working with.

I. PCR I (performed with inexpensive non-high-fidelity Taq)

NEB One- Taq 2x Master Mix 8.0 microliters 88.0 microliters
Forward Primer (10mM) 1-X 0.40 microliters 4.40 microliters
Reverse Primer (10mM) 2-6 0.40 microliters 4.40 microliters
Pure H2O 6.2 microliters 68.2 microliters
Master Mix Total 15 microliters 165 microliters

PCR was run using PCR1 on BIORAD #1/2

  • 94 degrees Celsius (30 sec)
  • 60 degrees Celsius (30 sec)
  • 68 degrees Celsius (45 sec)

Then it was put on a 4-10 degree Celsius infinite hold. The products were run on a 1.5% agarose gel with a 100 bp ladder at 130V for 40 minutes

[Successful amplification results in a. smear of fragments typically ranging from 50-700 base pairs] Ours was successful and was documented by Prof. Paul

Since the test was successful, we moved on to the final PCR reaction

II. Final PCR

Next we ran the final PCR. We made a different master mix for this part.

Phusion DNA Polymerase 0.31 microliters 3.41 microliters
5X Phusion HF buffer 6.25 microliters 68.75 microliters à 69
Forward Primer (10mM) 1.56 microliters 17.16 microliters à 17.2
Reverse Primer (10mM) 1.56 microliters 17.16 microliters à 17.2
DNTPs (10mM) 0.63 microliters 6.93 microliters
DMSO 0.94 microliters 10.34 microliters à 10.3
Pure H2O 10.75 microliters 118.25 microliters à 118
Master Mix Total 22.0 microliters 242.04 microliters

NOTE: We were assigned the PCR 2_1 for our master mix (assigned by Paul). For some reason we ran out of master mix when we were putting it in the samples so I (mistakingly) suggested that we use the next table’s master mix. They gave it to us and we used it BUT they had a different reverse primer :/ [they had 2_7]. The wrong master mix was used in reaction 21 and 22.

We ran this the same way and under the same conditions as the test PCR run (written above)

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November 12 – ddRAD-seq + adapter ligation

In this lab, we digested and ligated our DNA samples.

I. DOUBLE DIGEST

For this part of the lab, we first double digested 100-1000 ng of high quality genomic DNA with restriction enzymes then used a digestion buffer appropriate for the two enzymes. We placed 6 microliter of each sample’s DNA in a PCR plate/tube and stored it on ice.

Then, we made a master mix for around 10-11 samples to make sure we had enough for our 8 reactions. We mixed it well and then stored it on ice.

CutSmart Buffer 10x 0.90 microliters 9.90 microliters
EcoRI-HF enzyme 0.28 microliters 3.08 microliters
MSPI enzyme 0.12 microliters 1.32 microliters
Pure H2O 1.70 microliters 18.7 microliters
Master Mix total 3.00 microliters 33.0 microliters

Next, we added 3 microliter of master mix into each sample.

  • the total reaction volume at this point was 9 microliter. We sealed the samples, vortexes them, centrifuged, and finally incubated them at 37 degrees Celsius for 8 hours on a thermocycler with a heated lid set to 50 degrees.

Our samples names and numbers from Alec (grad student who collected these samples) are shown below-

DIRA009 14
INVR002 22
SCHO002 26
DIRA006 11
MONO006 10
CHIM005 5
PRBN002 17
TROS002 28

II. ADAPTER LIGATION

The next part of this lab was the adapter ligation. We thawed the working stock EcoRI and Maple adapters made previously in the Paul Lab. The P1 EcoRI adapters are as follows.

PAUL LAB ID Adapter name
Eco_2 AACCA_EcoRI
Eco_3 CGATC_ EcoRI
Eco_4 TCGAT_ EcoRI
Eco_5 TGCAT_ EcoRI
Eco_6 CAACC_ EcoRI
Eco_7 GGTTG_ EcoRI
Eco_8 AAGGA_ EcoRI
Eco_9 AGCTA_ EcoRI
Eco_10 ACACA_ EcoRI

Our table was assigned 17-24 as our sample ID’s so we used Eco Adapters 2-9 respectively- those are shown below as well.

SAMPLE ID ECO ADAPTER
1 (17) 2
2 (18) 3
3 (19) 4
4 (20) 5
5 (21) 6
6 (22) 7
7 (23) 8
8 (24) 9

We added one microliter of each respective EcoRI adapter to the digested DNA, then made the master mix. Again, we made 11 reactions worth of master mix (130%).

CutSmart buffer 10x 0.4 microliters 4.4 microliters
ATP (10mM) 1.30 microliters 14.3 microliters
T4 ligase 0.20 microliters 2.2 microliters
Pure H2O 0.10 microliters 1.1 microliters
Universal P2 MspI adapter 1.00 microliters 11.0 microliters
Master Mix Total 3.00 microliters 33.0 microliters

We added three microliters of this second master mix to the digested DNA

  • The total reaction volume is now 13 microliter. Samples were sealed, vortexed, centrifuged, and incubated at 16 degrees Celsius for 6 hours on a thermocycler with a heated lid set to 50 degrees Celsius.
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November 5 – PCR Reactions (Mimulus gutatus)

This week we ran the PCR reactions for our Mimulus guttatus project – materials shown below. We made enough Mastermix for 18 reactions even though we only had 12- just to make sure we had enough.

Ingredients Per Reaction [18 reactions]
ddH2O 13.36 240.48
10x buffer 2.00 36
MgCl2 2.00 36
BSA 1.00 18
dNTPs 0.20 3.6
F-primer 0.20 3.6
R-primer 0.20 3.6
Taq 0.04 .72 àRounded to .80 (for pipette)
Template 1.00 n/a
Total 20.00  

We labeled our tubes as shown below:

Tubes Specimen ID
QS 1 MM MM1
QS 2 MM MM2
QS 3 MM MM3
QS 4 OY OY01
QS 5 OY OY02
QS 6 OY OY03
QS 7 EB
QS 8 RDRK001
QS 9 SHOR005
QS 10 KRS1
QS 11 KRS2
QS 12 KRS3

We added 19 microliter of Mastermix to each of the 12 reactions, mixed them on the vortex then Prof. Paul ran the PCR reaction for us.