JULY

Tuesday, July 1

Ran instead the PCR Thermalcycler Program for Gibson Assembly Protocol; still used the same 50 μL reaction mixture. Ran gel to verify results with a 2 log DNA ladder. Still unsuccessful.

Wednesday, July 2 - Tuesday, July 8

Redesigned primers 3 and 4 for pMBI operon in the hopes of fixing the problem with PCR success. Designed primers for pMevT operon and ordered. Researched and decided to produce zeatin as final high-value product goal.

Wednesday, July 9

Reconsistuted and ran PCR program on primers for PMK. Ran gel electrophoresis to confirm success. Digested puc19 as specified in Gibson Assembly protocol.

Reaction Mixture for puc19 Digestion
Component	Volume (μL)
PCR product	45
NEB 10X Cutsmart Buffer	6
Nuclease-free Water	8
DpnI	1
Total	60

Incubated at 37 °C for 1 hour, then transferred to fridge.

Thursday, July 10

Followed Roche PCR cleanup protocol for successful pMBI PCR product. Used nanodrop (Thermo-Fischer nanodrop 2000) to analyze the concentration of our purified PCR products.

Initial Concentrations
Sample	Concentration (ng/μL)
MK	7.8
PMK a	3.7
PMK b	1.5
MVD1	5.2
idi	17.0
puc19	15.2

These concentrations were too low to complete the Gibson reaction, so we dried samples (concentrated) using a SAVANT.

Final Concentrations
Sample	Concentration (ng/μL)
MK	31.4
PMK a	112.7
PMK b	11.9
MVD1	47.6
idi	42.8
puc19	50.2

Figured out appropriate volumes to add of each component to run Gibson reaction.

• Used Equation 1 to find pmol/μL of DNA
Fragment concentration (ng/μL) = X (pmol/μL) x fragment length (bp) x 650 (daltons) / 1000 (Equation 1)
• Used Excel program to determine a value of pmol of total DNA (between 0.2 and 1.0 pmol) that had a volume of ~5 μL.

Mixture for 0.3 pmol of total DNA (0.06 pmol of each sample)
Sample Name	Concentration (ng/μL)	Molar Concentration (pmol/μL)	Fragment Length (bp)	Volume for Mixture (μL)
MK	31.4	0.0363	1330	1.65
PMK a	112.7	0.128	1354	0.47
MVD1	47.6	0.0614	1191	0.98
idi	42.8	0.121	543	0.49
puc19	50.2	0.0299	2585	2.00
Total	-	-	7003	5.59

Friday, July 11

Ran Gibson reaction and transformed cells immediately as per protocol. Left overnight in incubator to culture.

Monday, July 14

Reconstituted pMevT primers and ran PCR (same 50 μL reaction). Our overnight cultures produced lawns but we suspect that this was due to contamination and not due to the success of the Gibson assembly. Streaked for isolation (streaked colonies from the lawn to try and produce isolated colonies) and left incubating overnight at 37° C. Also made new LB + Agar + Amp¹⁰⁰ plates and autoclaved all pipette tips and glass beads used for plating and transformation. Left test plate in incubator overnight as well to see if plates were contaminated.

Tuesday, July 15

Test plate did not result in any growth but as fungi was present on one of the sleeves, we disposed of the plates anyway. Ran gel on pMevT PCR products, all unsuccessful except for atoB product.

Wednesday, July 16

Ran PCR with Thermalcycler Program from Gibson Assembly using 57° C for second annealing temperature. Ran gel to confirm results, all negative. With new plates, ran same Gibson reaction and transformed and incubated cells for overnight culturing.

Thursday, July 17

Successful growth on both Gibson and control plates. We suspect that this is due to puc19 ligating to itself, as there were more colonies on the control plate than on the Gibson plate. Continued with protocol to verify suspicion and ran colony PCR on the colonies from the Gibson reaction. Ran gel to confirm PCR results; all negative.

Tuesday, July 22

Retried pMevT PCR using a gradient on the thermalcycler machine. Also used new 50 μL reaction mix.

50 μL PCR Reaction Mixture
Component	Volume (μL)	Concentration (μM)
5X Phusion Buffer	10	1X
10 mM dNTPs	1	200
Primer A	2.5	0.5
Primer B	2.5	0.5
Template DNA	~1	-
Phusion DNA Polymerase	0.5	0.2 U/μM
Nuclease-Free Water	Fill to 50 μL	-
Total	50	-

PCR Thermalcycler Program was run as specified in Gibson Assembly protocol with a gradient with 56 °C, 65 °C, and 72 °C as temperatures for 2^nd annealing step; ran three reactions (one for each temperature). Ran gel to confirm results, was able to amplify all four pieces successfully.

Wednesday, July 23

Digested all pMevT PCR products as per suggestion from Dr. Peebles to ensure no false positives from Gibson. Incubated digestion mix at 37 °C for over 1 hour. Followed PCR Purification protocol for all tubes.

Thursday, July 24

Checked the rest of the colonies with colony PCR products from pMBI on gel, all results negative.

Monday, July 28

Redigested idi PCR product for pMBI. Ran 50 MICOL reaction instead of 100 MICROL. Incubated at 37 °C for over an hour. Used nanodrop to figure out concentrations (see Thursday, July 10 for use instructions).

Initial Concentrations
Sample	Concentration (ng/μL)
AtoB	118.8
HMGS a	2.7
HMGS b	2.0
tHMG	2.6
puc19 a	9.9
puc19 b	6.3

Tuesday, July 29

Dried samples completely with SAVANT. Going to resuspend all samples to be at a concentration of 50 ng/MICROL in Elution Buffer from our PCR cleanup kit.

Final Concentrations
Sample	Concentration (ng/μL)	Resuspension Volume (μL)
atoB	50.0	237.6
HMGS a	50.0	5.4
HMGS b	50.0	4.0
tHMG	50.0	5.2
puc19 a	50.0	19.8
puc19 b	50.0	12.6

Wednesday, July 30

Ran PCR on PMBI with gradient (55 +/- 3.5 °C); used an extension time of 3 minutes and left overnight to run.

Thursday, July 31

Ran gel electrophoresis on PCR products for pMBI. Was able to amplify tHMG and puc19 for both temperatures. Made another set of LB + Amp¹⁰⁰ plates.

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