July

(07/18/16)

PCR Amplification (SFR2, tSFR2, desA) with Phusion High Fidelity Polymerase

1. Mix the following reagents for PCR amplification, each reaction of 50 µl and we did one negative control where we did not put the template DNA. We also use 2 different buffers because we figured that our gene sequence has a high GC%. Master-mix reaction mixture (4X):

• Nuclease free H2O 134 µl
• 5X HF/GC buffer 40 µl
• DMSO 2 µl
• dNTPs 4 µl
• Forward primer (10 µM) 5 µl
• Reverse primer (10 µM) 5 µl
• Template DNA 6 ng (2 ng/reaction)
• Phusion Polymerase 2 µl

2. Run the PCR reaction with touch up program, where we run 7 cycles with the temperature of the primer without the overhang and another 30 cycles with the temperature of the primer full sequence.

• SFR2 Touch-up PCR Program

Step 1	Step 2	Step 3
98°C 45 secs (1 cycle)	98°C, 61.7°C, 72°C 7 secs, 20 secs, 80 secs (7 cycles)	72°C 7 mins (1 cycle)
98°C 45 secs (1 cycle)	95°C, 71.3°C, 72°C 7 secs, 20 secs, 80 secs (30 cycles)	72°C 7 mins (1 cycle)

• tSFR2 Touch-up PCR Program

Step 1	Step 2	Step 3
98°C 45 secs (1 cycle)	98°C, 61.7°C, 72°C 7 secs, 20 secs, 80 secs (7 cycles)	72°C 7 mins (1 cycle)
98°C 45 secs (1 cycle)	95°C, 72°C, 72°C 7 secs, 20 secs, 80 secs (30 cycles)	72°C 7 mins (1 cycle)

• desA Touch-up PCR Program

Step 1	Step 2	Step 3
98°C 45 secs (1 cycle)	98°C, 62.9°C, 72°C 7 secs, 20 secs, 60 secs (7 cycles)	72°C 7 mins (1 cycle)
98°C 45 secs (1 cycle)	95°C, 72°C, 72°C 7 secs, 20 secs, 60 secs (30 cycles)	72°C 7 mins (1 cycle)

3. Run Gel Electrophoresis with 0.8% agarose at 150 V for 30 minutes. 1 KB plus ladder is used.

• A-C: SFR2 with HF buffer
• D : SFR2 negative control
• E-G: SFR2 with GC buffer
• H : SFR2 negative control
• I-K: tSFR2 with HF buffer
• L : tSFR2 negative control
• M-O: tSFR2 with GC buffer
• P : SFR2 negative control

• A-C: desA with HF buffer
• D-F: desA with GC buffer

4. Gel Extraction was done with Wizard SV Gel and PCR clean up kit by Promega. Then, DNA concentrations were measured with nano drop.

(07/19/16)

Restriction Digest of Riboswitch Plasmid with EcoRI and XbaI

1. Restriction digest for the riboswitch plasmid (3745 bp) with Fast Digest from NEB in total reaction of 20 ul. Set up the following reaction:

• Fast Digest buffer 2 µl
• EcoRI restriction enzyme 1 µl
• XbaI restriction enzyme 1 µl
• Plasmid 1 µg
• Nuclease free H2O until 20 µl

2. Run Gel Electrophoresis with 0.8% agarose at 150 V for 30 minutes. Gel extraction and concentration is measured with nano drop. Plasmid Concentration: 8.1 ng/µl

Gibson Assembly

1. Calculate the number of pmols needed for each fragment if we use 30 ng of the vectors.

(30ng X 1000)/(3745bp X 650)= 0.012 pmols

2. We used 3 fold of excess insert, so the number of pmols for plasmid will be 3 times the amount of insert we need (0.036 pmols).

Amount of SFR2 needed:
(x X 1000)/(1869bp X 650)= 0.036 pmols
x= 44.91 ng

Amount of tSFR2 needed:
(x X 1000)/(1788bp X 650)= 0.036 pmols
x= 41.84 ng

Amount of desA needed:
(x X 1000)/(1095bp X 650)= 0.036 pmols
x= 25.62 ng

3. Gibson Assembly master mix from NEB is used, so all we need to add is the plasmid backbone and the inserts according to the amount we calculated above in total reaction of 20 µl. We also run negative control where we just put the plasmid backbone without insert.

4. Set up the reaction in 50°C for 30 minutes

Transformation of the Recombinant Plasmid into DH5α Competent Cells

1. Thaw competent cells on ice for around 5-10 minutes.

2. Use 2 different volumes of Gibson product to mix with the competent cells. Add each tube of competent cells (50 µl) with 2.5 µl and 5 µl to 50 µl.

3. Incubate on ice for 20 minutes. Heat shock cells at 42°C for 45 seconds.

4. Put it back on ice for another 2 minutes and add 900 µl of LB into the tube. Incubate in the 37°C shaker for an hour.

5. Spread plate 50 µl and 200 µl of them onto LB plates with spectinomycin antibiotic as the selective marker.

6. Incubate at 37°C incubator overnight

Result: No colony grew in SFR2 and tSFR2 plates, but there were several colonies grew in the desA plate.

(07/20/16)

Prepare Bacterial Culture for Mini Prep

1. Add 50 ul of spectinomycin antiobiotic into 50 ml LB

2. In culture tubes, take 4 ml of LB + spectinomycin media. Pick isolated colonies formed on the desA transformation plate and mix well with the media (pipette up and down).

3. Incubate overnight at 37°C shaker.

(07/21/16)

Plasmid Purification (Miniprep)

1. Spin down overnight culture in microcentrifuge tubes and discard the supernatant.

2. Re-suspend each pellet in 215 µl buffer P1 (cell resuspension solution).

3. Add 215 µl buffer P2 (cell lysis solution) and invert the tube 8 times to mix.

4. Add 330µl buffer P3 and invert 8 times to mix.

5. Spin down at 16,000 g for 5 minutes.

6. Apply 730 µl of supernatant to DNA column.

7. Spin down at 16,000 g for 1 minute and discard flow through.

8. Wash column with 700 µl wash buffer and spin down at 16,000 g for another minute. Discard flow through. Wash one more time with 500 µl wash buffer and discard flow through.

9. Dry spin column for 2 minutes to evaporate all the ethanol.

10.Elute into new microcentrifuge tube with 37 µl nuclease free H₂O.

11. Measure concentration with nano drop.

• SFR2 recombinant plasmid: 330.3 ng/ul
• tSFR2 recombinant plasmid: 368.8 ng/ul
• desA recombinant plasmid: 453.3 ng/ul

(07/22/16)

Recombinant Plasmid Verification with Restriction Digest

1. Run virtual digest of desA recombinant plasmid with KpnI restriction enzyme and SFR2, tSFR2 recombinant plasmid with SalI.

1 KB plus ladder
Lane 1: desA recombinant plasmid
Lane 2: plasmid only
Lane 3: SFR2 recombinant plasmid
Lane 4: tSFR2 recombinant plasmid
Lane 5: plasmid only

2. Make the restriction reaction mixture in a total volume of 20 µl. Set up the following reaction:

• 1 ug plasmid DNA
• 2 ul cutsmart buffer
• 1 ul restriction enzyme
• Nuclease free H2O until 20 ul

3. Run gel electrophoresis in 0.8% agarose at 150 V for 30 minutes.

We got positive result, where the total of 3 bands showed up at the correct size
for desA recombinant plasmid and 2 bands for the control (plasmid only).

We got negative results, it did not seems like the one in virtual digest.
All the bands showed up almost at the same position as the negative control (plasmid only).

(07/28/16)

SFR2 & tSFR2 Plasmid Verification with Restriction Digest
(2^nd attempt)

1. Run virtual digest of SFR2 and tSFR2 recombinant plasmid with XhoI and KpnI.

1 KB plus ladder
Lane 1: SFR2 recombinant plasmid
Lane 2: tSFR2 recombinant plasmid
Lane 3: plasmid only

2. Make the restriction reaction mixture in a total volume of 20 µl. Set up the following reaction:

• 1 ug plasmid DNA
• 2 µl cutsmart buffer
• 1 µl XhoI
• 1 µl KpnI
• Nuclease free H2O until 20 ul

3. Run gel electrophoresis in 0.8% agarose at 150 V for 30 minutes.

We got negative results. All the bands still showed up exactly like the negative control
(plasmid only), where the top band showed up around 2 kb instead of 4 kb.

(07/29/16)

Send desA recombinant plasmid to sequencing facility

1. Dilute recombinant plasmid to 50 ng/µl.

2. In 1.5 ml centrifuge tube, take 5 µl of diluted recombinant plasmid, total of 250 ng, and 5 µl of forward or reverse sequencing primer, total of 25 µM.

3. Plasmid is ready to be sequenced!

August

(08/03/16)

Analyze the sequencing result of SFR2, tSFR2, and desA

1. Open Benchling software and clone the SFR2, tSFR2 and desA to the riboswitch plasmid to prepare for sequence alignment.

2. Open the sequencing file (.ab1) in Snapgene Viewer and copy the whole sequence.

3. In Benchling, open the recombinant plasmid sequence, choose alignment button on the right side of the screen and paste the sequence we copied from Snapgene viewer into the sequence box.

4. Analyze how the gene sequence align with the expected recombinant plasmid.

5. Our desA plasmid sequence looks really well-aligned with the expected recombinant plasmid, but we lost a big chunk of sequence in our SFR2 and tSFR2 gene sequence.

Team:MSU-Michigan/Notebook.html

Michigan State University iGEM 2016

July

PCR Amplification (SFR2, tSFR2, desA) with Phusion High Fidelity Polymerase