Notebook
October 13, 2016
Transformed 5uL of BBa_J04450 at a concentration of 20pg/uL into Stellar competent cells using the following protocol:
- Remove competent cells from -80°C freezer and thaw on wet ice (10-15 minutes).
- Add DNA (pre-chilled) to 100μL of cells on ice. Stir briefly with a pipet tip – DO NOT pipet up and down to mix as this can heat up cells and introduce bubbles.
- Incubate on ice for 30 minutes.
- Heat-shock cells by placing tube in a 37°C heat block for 1 min.
- Return cells to ice for 2 minutes.
- Add 950μL of room temperature SOC to the cells in the culture tube.
- Place tubes in a shaking incubator at 250 rpm for 1 hour at 37°C.
- Centrifuge at 1,000g for 1 min, remove and discard 950μL of supernatant, resuspending cells gently in remaining SOC (~150μL).
- Plate entire sample of transformed cells on LB agar plates with the chloramphenicol.
- Incubate plates upside down overnight at 37°C.
October 14, 2016
Colonies from previous transformation were selected on the criteria of size and isolation for overnight culture. The selected colonies were removed with a pipette tip. The pipette tip was then placed into a 14mL culture tubes containing 10mL LB and 10uL 1000X Chloramphenicol. The plates were incubated overnight at 37°C.
October 15, 2016
Verification of BBa_J04450 via PCR
Master Mix
20µL Phusion Buffer
2µL Primers
2µL dNTP
Primer Dilution
1 µL SB-Prep Forward Primer
1 µL SB-Prep Reverse Primer
2 µL PCR Grade H2O
Setup 2 50µL Reactions:
PCR A
1µL DNA (from mini prep of overnight culture A)
12µL Master Mix
0.5 µL Phusion
36.5µL PCR Grade H2O
PCR B
2µL DNA (from mini prep of overnight culture B)
12µL Master Mix
0.5 µL Phusion
35.5 PCR grade H2O
Amplify with the following program:
98°C for 30s
35 Cycles of:
98°C for 10s
47-65°C for 30s
72°C for 30-45s/1kb
72°C for 10 minutes
12°C hold
PCR Products were analyzed using the gel electrophoresis protocol that follows on October 16, 2016.
October 16, 2016
Digest of Vector and Insert
Set up 2 20uL reactions:
Vector
5µL 10X Cutsmart Buffer
1µL NotI
14µL vector DNA
17µL ddH2O
Insert
5µL 10X Cutsmart Buffer
1µL NotI
29.2µL trkH DNA
14.9µL ddH2O
Incubate in thermocycler using the following program:
- Incubate in thermocycler as follows:
- 37°C for 2 hours
- 12°C hold
- Add 1μL Calf Intestinal Phosphatase to the vector reaction only and incubate at 37°C for 1 hour.
Digest products were analyzed using the following protocol:
Gel Electrophoresis Protocol
- Add to 250mL Erlenmeyer flask
- 0.5g Agarose
- 50mL TAE
- Heat in microwave until completely dissolved. Do not allow liquid to boil over, remove and swirl periodically.
- Add 5μL Gel Green
- Pour into gel case (make sure to eliminate bubbles with pipette tip) and insert comb. Allow gel to set. Can be left at 4°C to accelerate setting, or stored (wrapped in plastic) for up to 1 week.
- Place gel into gel box and cover with TAE.
- Add 10uL 6X loading dye to each PCR reaction and load 12uL of each.
- Load 10μL TriDye 2-Log Ladder to the first lane.
- Run until dye front reaches the end of the gel at 100V (about 75 min).
- Image on ChemiDoc using either Ethidium Bromide or Gel Green program.
The gel was loaded as follows
- 10uL TriDye 2-Log Ladder
- Lanes 2-4 17uL Digested Insert
- Lanes 6-8 17uL Digested Vector
The bands of interest were excised based on size and purified using the following protocol.
Gel Purification of Digest
- Weigh gel slice.
- Place a SV Minicolumn into a collection tube.
- Add Membrane Binding Solution at a ratio of 10μl of solution per 10mg of agarose gel slice.
- Vortex (or invert) the mixture and incubate at 55°C for 10 minutes or until the gel slice is completely dissolved.
- Transfer up to 350μL dissolved gel sample to column and incubate 3 min at room temperature.
- Centrifuge 16,000g for 1 min and discard flowthrough.
- Add 700μL Membrane Wash solution, centrifuge 16,000g for 1 min and discard flowthrough.
- Perform second was with 500μL Membrane Wash solution, centrifuge 16,000g for 5 min and discard flowthrough.
- Dry membrane by centrifuging 16,000g for 1 min.
- Transfer column to new, sterile 1.5mL tube and add 23.5μL Nuclease-free water. Incubate at room temperature for 4 min.
- Centrifuge at 16,000g for 1 min.
- Determine DNA concentration by NanoDrop.
DpnI Digest of BBa_J04450 for Fast Cloning
1µL DpnI
2µL Cutsmart Buffer
15µL PCR products (*PCR products not cleaned)
2µL ddH2O
Incubate in the thermocycler with the following program:
37C for 2hrs
80C for 20 mins
4C Forever
Fast Cloning
DpnI Digest of BBa_J04450 and trkH were transformed into stellar cells using the aforementioned transformation protocol in the following volumetric ratios:
1uL vector : 1uL insert
1uL vector : 2uL insert
1uL vector : 3uL insert
October 17, 2016
Ligation of Vector and Insert
Set up 1 50µL reaction:
5µL 10X T4 ligase buffer
20µL Insert
6.19µL Vector
1µL DNA Ligase
17.81µL ddH2O
Incubate in the thermocycler with the following protocol:
45oC 5 mins
16oC overnight
24oC 30 mins
Overnight Culture
Colonies from the fast cloning transformation were selected from each of the different dilutions. Overnight cultures were inoculated using the aforementioned culture protocol.
October 18, 2016
Verification of Insert PCR
Master Mix
30µL Buffer
3µL Primers
3µL dNTP
Primer Dilution
2 µL VF2 Primer
2 µL VR Primer
4 µL PCR Grade H2O
All DNA was diluted to 50ng/uL
Setup 3 50µL Reactions:
PCR 1:1 Dilution
1µL DNA
12µL Master Mix
0.5 µL Phusion
36.5µL PCR Grade H2O
PCR 1:2 Dilution
2µL DNA
12µL Master Mix
0.5 µL Phusion
36.5 PCR grade H2O
PCR 1:3 Dilution
1µL DNA
12µL Master Mix
0.5 µL Phusion
36.5µL PCR Grade H2O
Amplify with the following program:
98°C for 30s
35 Cycles of:
98°C for 10s
47-65°C for 30s
72°C for 30-45s/1kb
72°C for 10 minutes
12°C hold
PCR Products were analyzed using the aforementioned gel electrophoresis protocol. The gel was loaded with 12uL of each sample and 10uL of ladder.
- Ladder
- 1:1 Dilution
- 1:2 Dilution
- 1:3 Dilution
Transformation of Ligation Products
The ligation products were transformed into Stellar cells at a volume of 20uL using the aforementioned transformation protocol.
