Team:OU Norman/Project/Notebook/meredith

6/25/14 - 3A Assembly of promoters and ADH6

Past two attempts were not successful. Again, this time we will increase amount of ADH6, to double it to 1.2ul. Additionally, in the ligation step we add the PCR water, buffer, 3 parts and lastly the T4 Ligase.

Before beginning:
Preheat the heat block and heat bath
Cross link PCR water
Have Tetracycline plates
Psi broth
Ice block
Retrieve enzymes from freezer: EcoR1, Spe1, Xba1, Pst1
Primers
BSA

Adh6#1	1000ng/755.6ng = 1.32ul
20A#14	500ng/316.5ng = 1.58ul
20E#14	500ng/198.7ng = 2.52ul
23B#13	500ng/405.8ng = 1.23ul
Psb1T3#1	500ng/178.9ng = 2.79ul

-Digestion: Combine in 1.5 Microcentrifuge Tube (add from bottom to top)

Promoter: 20A#14

Part	Volume(uL)
Promoter	1.58
EcoR1	1
Spe1	1
10x Buffer	5
BSA	1
PCR water	40.42

Promoter: 23B#13

Part	Volume(uL)
Promoter	1.23
EcoR1	1
Spe1	1
10x Buffer	5
BSA	1
PCR water	40.77

Promoter: 20E#14

Part	Volume(uL)
Promoter	2.52
EcoR1	1
Spe1	1
10x Buffer	5
BSA	1
PCR water	39.48

Downstream Gene: Adh6#1

Part	Volume(uL)
Gene	1.32
EcoR1	1
Spe1	1
10x Buffer	5
BSA	1
PCR water	40.68

Plasmid: pSB1T3

Part	Volume(uL)
Plasmid	2.79
EcoR1	1
Spe1	1
10x Buffer	5
BSA	1
PCR water	39.20

-Place in heat bath for 45 mins at 42C
-Heat-kill enzymes on heat block fro 15 mins at 80C

Ligation:
-Combine in 1.5 microcentrifuge tube
-4ul of upstream part (promoters: 20A#14, 20E#14, or 23B#13)
-4ul of downstream part (Adh6)
-4ul of destination plasmid (PsB1T3)
-1ul of T4 Ligase (Add Last)
-4ul of 10x T4 Ligase buffer
-22ul of H2O (Add First)
-Incubate (3 tubes) on the bench top for 20 mins
-Prepare E. coli cells in ice to thaw for transformation

Transformation:
-Combine in 1.5 microcentrifuge tube
-Transform 4ul of ligation product into 100ul of competent Top 10 E. coli cells for each transformation
-Ice for 30 mins
-Heat-shock in heat bath for 60 seconds at 42C
-Place back on ice for 5 mins
-Add 600ul of Psi broth to each tube
-Incubate at 37C for 2 hours with 200rpm shaking

Plating:
-Streak each transformation product onto individual plates
-6 plates (2 for each promoter combination)
-Each Promoter combination will be diluted 1:1 and 1:10
-The 1:1 dilution is already in tube – plate ~90ul
-Add 10ul to 90ul of Psi broth for 1:10, plate ~90ul
-Allow to incubate for 36-48 hours.

3A assembly was not successful, no growth.

7/9/14 –Transformation of Adh6, Ter and KIVD

-Retrieve each gene
-Retrieve competent cells
-Psi Broth
-using Adh6#1 = 755.6ng/ul
-Trans-2-enolase (TER)#8 = 793ng/ul
-Transform 4ul of gene into 100ul of Top 10 E.coli cells
-Ice for 30 mins
-Heat shock in heat bath for 1 min at 42C
-Ice for 5 mins
-Add 600ul of Psi broth to each tube
-Incubate at 37C for 2hours with 200rpm shaking
-Plate each product on separate plates
-1:1 dilution straight from tube – plate ~90ul
-1:10, add 10ul to 90ul of Psi broth – plate ~90ul
-Allow to incubate 24-48 hours at 37C, monitor for growth, do DNA Extraction

7/17/14 – DNA/Plasmid Extraction

-Extraction for Adh6, KIVD, 20A, 20E, 4P and 6D
-Using NEW DNA Extraction kit: GeneJet Plasmid Miniprep Kit (250)
-Need: 1.5 Microcentrifuge tubes, Centrifuge, fresh cultures, prep kit

Protocol:
1. Add 250ul of Resuspension buffer to a 1.5 microcentrifuge tube
2. Transfer 3 large loopfuls of culture from freshly incubated plate to tube with buffer
3. Use 1ml blue pipette tip to mix cells with buffer until homogenized
4. Add 250ul of Lysis Buffer, vortex until no clumps of cells are visible, let sit for 4 mins, but no longer than 4
5. Add 350ul of Neutralization buffer and mix thoroughly by vortex
6. Centrifuge for 5 mins at 13,000rpm
7. Set pipette to 800ul and extract as much of the supernatant as possible and add it to spin column from kit. Do not transfer debris.
8. Centrifuge for 1min
9.Discard flow-through
10. Bind the DNA to the filter in the spin column by adding 500ul of Wash Solution
11. Centrifuge for 1min
12. Discard flow-through
13. Repeat Step 10 with 500ul of Wash Solution
14. Centrifuge for 1 min
15. Discard flow-through
16. Centrifuge for extra minute to remove residual wash buffer
17. Place Spin column in 1.5 microcentrifuge tube
18. To elute DNA, add 50ul Elution Buffer or DI water to the center of the spin column, but do not touch the filter with the pipette tip.
19. Let stand for 2 mins.
20. Centrifuge for 2 mins
21. Discard spin column
22. The 1.5 microcentrifuge tube holds the plasmid/DNA in solution
23. Quantify via nanodrop

Quantification of KIVD with 4P or 6D, and 20A, 20E and Adh6

7/17/14 – 3A assembly of Promoter 23B with Adh6

Use 1500ng of Adh6 to try and yield more success

Before beginning:
-Preheat the heat block and heat bath
-Cross link PCR water
-Have Tetracycline plates
-Psi broth
-Ice block
-Retrieve enzymes from freezer: EcoR1, Spe1, Xba1, Pst1
-Primers
-BSA

Adh6#3	1500ng/628.4ng = 2.39ul
23B#3	500ng/115.4ng = 4.33ul
PsB1T3#1	500ng/178.7ng = 2.80ng

Digestion: Combine in 1.5 Microcentrifuge Tube (add from bottom to top)

Promoter: 23B#3

37.67

Part	Volume (uL)
Promoter	4.33
EcoR1	1
Spe1	1
10x Buffer	5
BSA	1
PCR water

Downstream Gene: Adh6#3

Part	Volume (uL)
Promoter	2.39
EcoR1	1
Spe1	1
10x Buffer	5
BSA	1
PCR water	39.61

Plasmid: pSB1T3

Part	Volume (uL)
Plasmid	2.80
EcoR1	1
Spe1	1
10x Buffer	5
BSA	1
PCR water	39.20

-Place in heat bath for 45 mins at 42C
-Neutralize enzymes on heat block for 15 mins at 80C

Ligation:
-Combine in 1.5 microcentrifuge tube
-4ul of upstream part (promoter 23B#3)
-4ul of downstream part (Adh6)
-4ul of destination plasmid (PsB1T3)
-1ul of T4 Ligase (Add Last)
-4ul of 10x T4 Ligase buffer
-22ul of H2O (Add First)
-Incubate on the bench top for 20 mins
-Prepare E. coli cells in ice to thaw for transformation

Transformation:
-Combine in 1.5 microcentrifuge tube
-Transform 4ul of ligation product into 100ul of competent Top 10 E. coli cells for each transformation
-Ice for 30 mins
-Heat-shock in heat bath for 60 seconds at 42C
-Place back on ice for 5 mins
-Add 600ul of Psi broth to each tube
-Incubate at 37C for 2 hours with 200rpm shaking

Plating:
-Streak each transformation product onto individual plates
-Each will be diluted 1:1 and 1:10
-The 1:1 dilution is already in tube – plate ~90ul
-Add ul to 90ul of Psi broth for 1:10, plate ~90ul
-Incubate at 37C and monitor for growth

Successful Colonies grown and libraries made on 7/18/14

7/17/14 – Media prep with Promoter, Adh6 in PsB1T3 in 2XYT media

We have had difficulty with the efficacy of our promoters (23B, 20A, 20E). To try and increase our yield we will attempt to increase the cell mass by growing the cells in an Erlenmeyer flask with a rubber stopper inside, a foam stopper and foil to cover.

-Add 75ul of 2XYT broth to a 250ml Erlenmeyer flask (this amount helps maintain aerobic environment within the broth and prevents it from boiling over in the autoclave)
-Autoclave
-Add cells (either 20A or 20E)
-Allow to incubate over night in 37C with 200-300rpm shaking
-Extract DNA (tomorrow)

7/18/14 – DNA Extraction of Promoters (23B, 20A, 20E) with Adh6

23B#3 made via 3A Assembly with Adh6 on 7/17/14

20A#15 and 20E#11 made yesterday in flask with Adh6 on 7/17/14

Materials:
-GeneJet Plasmid Miniprep Kit (250)
1.5 Microcentrifuge tubes
-Centrifuge
-Fresh Cultures (broth or plate) (3-4 loopfuls)

Protocol:
1. Add 250ul of Resuspension buffer to a 1.5 microcentrifuge tube
2. Transfer 3 large loopfuls of culture from freshly incubated plate to tube with buffer
3. Use 1ml blue pipette tip to mix cells with buffer until homogenized
4. Add 250ul of Lysis Buffer, vortex until no clumps of cells are visible, let sit for 4 mins but no longer than 4
5. Add 350ul of Neutralization buffer and mix thoroughly by vortex
6. Centrifuge for 5 mins at 13,000rpm to pellet debris and chromosomal DNA
7. Set pipette to 800ul and extract as much of the supernatant as possible and add it to spin column from kit. Do not transfer debris.
8. Centrifuge for 1min
9.Discard flow-through
10. Bind the DNA to the filter in the spin column by adding 500ul of Wash Solution
11. Centrifuge for 1min
12. Discard flow-through
13. Repeat Step 10 with 500ul of Wash Solution
14. Centrifuge for 1 min
15. Discard flow-through
16. Centrifuge for extra minute to remove residual wash buffer
17. Place Spin column in 1.5 microcentrifuge tube
18. To elute DNA, add 50ul Elution Buffer or DI water to the center of the spin column, but do not touch the filter with the pipette tip.
19. Let stand for 2 mins.
20. Centrifuge for 2 mins
21. Discard spin column
22. The 1.5 microcentrifuge tube holds the plasmid/DNA in solution
23. Quantify via nanodrop - >130ng/ul desired

Results were insufficient:
20A#15 + Adh6 + PsB1T3 = 8.5 ng/ul
20E#11 + Adh6 + PsB1T3 = 16.7 ng/ul

7/18/14 – Colony PCR of KIVD, promoters and Adh6

Performed on KIVD + 4P or 6D and promoters (23B, 20A, 20E), and Adh6

30ul per reaction : 30ul x 30 reactions = 900ul total

Calculate total for extra 3 reactions just in case
-450ul master mix
-2ul Forward primer
-2ul Reverse primer
-446ul PCR water (total to 900ul)
Total = 900ul

PCR set up:
-5min at 94C
30 sec at 94C
1 min at 51C
3 min at 72C
10 min at 72C
Store at 4C

7/21/14 – Gel of PCR Products from 7/18/14

-Objective of the gel is to visualize which colonies contained the desired plasmid from previous 3A assemblies.

Materials: Agarose, 1x TAE buffer, Gel mold with electrodes, PCR product

1% gel: 0.4g agarose with 40ml of TAE Buffer = larger gel double amounts
-Combine in Erlenmeyer flask
-Microwave until clear, no chunks
-Pour into gel mold and allow to cool
-Voltage – small gel = 85 volts, large gel = 115 volts

In gel:
-in first well, load 5ul of KB ladder
-3ul dye in each sample well
-5ul DNA in each sample well

After gel ran, most of the DNA appeared to remain in the wells, even after running under higher voltage. Ultimately, there were no visible bands for promoters + Adh6. There was evidence for bands for KIVD + terminators.

7/23/14 – Transformation of P#1 and P#2 into DH5α with PIKM1

-Using previously assembled plasmid #1 (Ori + Pptb- +MLSR + 4P + PsB1C3) and Plasmid #2 (Ori + Pptb- +MLSR + 6D + PsB1C3) and DH5α with PIKM1 (methylating E.coli) to transform.

-Protocol:
-4ul of ligation product (P1#4 or P2#2) into 100ul of competent E. coli methylating strain cells, located in -80C freezer.
-Ice for 30 mins
-Heat shock for 1 min at 42C
-Ice for 5 mins
-Add 600ul of Psi Broth to tubes
-Incubate at 37C for 2 hours with 200rpm shaking
-Plate on Chloramphenicol/Kanamycin plates
-Allow to incubate for 24-48hours at 37C

Not successful, no colonies formed after 72 hours.