This lab we ran a test PCR to see if there was a successful library construction on our Mimulus guttatus samples using test PCR.

PCR I – Test Library Construction Success

NEB One-Taq 2x Master Mix – 80 uL

Forward Primer – 4 uL

Reverse Primer (PCR 026) – 4 uL

Pure H2O – 62UL

150 uL

Total reaction volume 16 uL

PCR1 ws used on BIORAD #1/2 at 94 degrees Celsius for 2 minutes, then 20 cycles of 94 degrees celsius for 30 seconds, 60 C fr 30 seconds, 68 C for 45 sec, and then at 4-10 C at infinite hold.

We then ran the products of PCR I for each sample on a 1.5% agarose gel with a 100 bp ladder at 130 V for 40 minutes.

PCR II – to generate final Illumina sequencing library

10 PCR reactions were set up in 25 uL volume.

Phusion DNA polymerase 3.1 uL

5x Phusion HF buffer 62.5 uL

Forward primer (PCR1_X) 15.6 uL

Reverse primer (2-7) 15.6 uL

DNTPS 6.3 uL

DMSO 9.4 uL

Pure H2O 107.5 uL

Master Mix Total 220 uL

Total reaction volume 25 uL

These were pipetted with individual samples into PCR tubes and then vortexed. PCR2 was run on BIORAD #2 for a number of cycles. Then 2 uL of the products of PCR2 were run on a 1% agrose gel with a 100 bp ladder.

Results of PCR 1 and PCR 2 are below.

 

This lab, we performed a DDRadseq of Mimulus guttatus samples. DNA samples collected from various locations (see Table 1 for codes) were chosen and 6 uL of each sample was placed in a PCR tube and stored on ice. A master mix was created with 9.9 uL of CutSmart 10x bugger, 3.1 uL ECORI-HF enzyme, 1.32 uL of MPSI enzyme, and 18.7 uL of pure H2O. 3 uL was added into each sample’s DNA. Samples were sealed, vortexed, and then placed in an incubator at 37 degrees Celsius for 8 hours on a thermocycler with a heated lid set to 50 degrees Celsius.

Our previous samples, prepared in the same way above, were then used to perform the second step of DDRADseq. Samples were placed on ice and 1 uL of EcoRI’s were added (see Table 2) to each sample. A master mix was created with 4.4 uL of CutSmart buffer 10x, 14.3 uL of ATP 10 mM, 2.2 uL of T4 igase enzyme, 1.1 uL pure H2O, and 11 uL of working stock universal P2 MspI adapter. 3 uL of this master mix was added to the digested DNA. Samples were sealed, centrifuged, and incubated at 16 degrees Celsius for 6 hours on a thermocycler with a heated lid set to 50 degrees Celsius.

Sample	Code
7	MONO 002
15	DIRA 010
24	LOTR 002
8	MONO 003
4	CHIM 004
16	PRBN 001

Table 1

Sample	Code
25	ECO_2
26	ECO_3
27	ECO_4
28	ECO_5
29	ECO_6
30	ECO_7
31	ECO_8
32	ECO_9

Table 2

This lab, we added microsatellites to the previously prepared samples of M. guttatus in order to process the DNA and to understand genetic diversity within M. guttatus.

Materials

• ddH2O
• 10x buffer
• MgCl2
• BSA
• dNTPs
• F-Primer (MIRI70)
• R-Primer (MIRI70)
• Taq
• Template
• Ice

11 PCR reactions were prepared to accommodate 9 samples of M. gutattus. All components of the PCR reaction were kept on ice. 146.9 uL of ddH2O, 22 ul of 10x bufffer, 22 ul of MgCl2, 11 uL of BSA, 2.2 uL of dNTPs, F-primer, and R-primer, and 0.44 uL of Taq were added to make approximately 19 uL per reaction. 1 uL of each sample was added to an individual tube and vortexed with the reaction mix to be processed.

This lab, we simply ran our gels for the samples processed in Lab 07.

12 loading dye dots of about 1 uL were pipetted onto a sheet of parafilm. 3 uL of each sample was placed into its own dot. All dots were loaded into the gel alongside others’ samples and run for approximately 40 minutes.