October 13,  2016

Transformed 5uL of BBa_J04450 at a concentration of 20pg/uL into Stellar competent cells using the following protocol:

1. Remove competent cells from -80°C freezer and thaw on wet ice (10-15 minutes).
2. 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.
3. Incubate on ice for 30 minutes.
4. Heat-shock cells by placing tube in a 37°C heat block for 1 min.
5. Return cells to ice for 2 minutes.
6. Add 950μL of room temperature SOC to the cells in the culture tube.
7. Place tubes in a shaking incubator at 250 rpm for 1 hour at 37°C.
8. Centrifuge at 1,000g for 1 min, remove and discard 950μL of supernatant, resuspending cells gently in remaining SOC (~150μL).
9. Plate entire sample of transformed cells on LB agar plates with the chloramphenicol.
10. 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:

1. Incubate in thermocycler as follows:         

1. 37°C for 2 hours  
2. 12°C hold

2. 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

1. Add to 250mL Erlenmeyer flask

1. 0.5g Agarose
2. 50mL TAE

2. Heat in microwave until completely dissolved.  Do not allow liquid to boil over, remove and swirl periodically.
3. Add 5μL Gel Green
4. 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.
5. Place gel into gel box and cover with TAE.
6. Add 10uL 6X loading dye to each PCR reaction and load 12uL of each.
7. Load 10μL TriDye 2-Log Ladder to the first lane.
8. Run until dye front reaches the end of the gel at 100V (about 75 min).
9. Image on ChemiDoc using either Ethidium Bromide or Gel Green program.

The gel was loaded as follows

1. 10uL TriDye 2-Log Ladder
2. Lanes 2-4 17uL Digested Insert
3. 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        

1. Weigh gel slice.
2. Place a SV Minicolumn into a collection tube.
3. Add Membrane Binding Solution at a ratio of 10μl of solution per 10mg of agarose gel slice.
4. Vortex (or invert) the mixture and incubate at 55°C for 10 minutes or until the gel slice is completely dissolved.
5. Transfer up to 350μL dissolved gel sample to column and incubate 3 min at room temperature.
6. Centrifuge 16,000g for 1 min and discard flowthrough.
7. Add 700μL Membrane Wash solution, centrifuge 16,000g for 1 min and discard flowthrough.
8. Perform second was with 500μL Membrane Wash solution, centrifuge 16,000g for 5 min and discard flowthrough.
9. Dry membrane by centrifuging 16,000g for 1 min.
10. Transfer column to new, sterile 1.5mL tube and add 23.5μL Nuclease-free water.  Incubate at room temperature for 4 min.
11. Centrifuge at 16,000g for 1 min.
12. 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.

1. Ladder
3. 1:1 Dilution
5. 1:2 Dilution
7. 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.