Tuesday, August 2nd
Tasks:
Michelle
- mCherry Gel Extractions
- mCherry 1 gel parts:
- 0.3712 g, dissolved in 1.1140 mL of GEX buffer
- 0.3501 g, dissolved in 1.0501 mL of GEX buffer
- mCherry 2 gel parts:
- 0.3139 g, dissolved in 0.9417 mL of GEX buffer
- 0.2385 g, dissolved in 0.7155 mL of GEX buffer
- 0.3169 g, dissolved in 0.9507 mL of GEX buffer
- mCherry 3 gel parts:
- 0.3782 g, dissolved in 1.1346 mL of GEX buffer
- 0.3702 g, dissolved in 1.1106 mL of GEX buffer
- 0.3138 g, dissolved in 0.9414 mL of GEX buffer
- mCherry 4 gel parts:
- 0.2850 g, dissolved in 0.855 mL of GEX buffer
- 0.3295 g, dissolved in 0.9885 mL of GEX buffer
- mCherry 5 gel parts:
- 0.3405 g, dissolved in 1.0215 mL of GEX buffer
- 0.3521 g, dissolved in 1.0563 mL of GEX buffer
- 0.3257 g, dissolved in 0.9771 mL of GEX buffer
- Used Epoch’s kit w/ team procedure modifications:
- Washed with WS twice
- Let stand for each wash for 1 minute
- Let stand before spinning for elution for 5 minutes
- Eluted in 20 μL of n.f. water instead of elution buffer
- Autoclaved 1 mL tips
- Autoclaved 250 mL of LB for Cam plates
- 0.25 mL of 1000X (35 mg/mL) Cam
- 1 sleeve poured
- HTML & CSS for the website
Paul
- Looked into DsbA as a SS for Cas9
- Transformed Gibson product:
- 50µL competent cells, 5 µL of Gibson product, 1µL of positive control (psb1c3-mrfp), 1µL of negative control (religation of tet backbone)
- -waited ~30 minutes
- 45 second heat shock
- 450µL of SOC added to each tube
- Incubated for an hour
- Plated 20 and 200µL plates for each sample
- Controls:
- Neg: Tet linearized BB+ligase 15 min (used 41 ng/uL BB)
- Pos: pSB1C3_J04450 (Transformation Efficiency Test kit: 50 pg/uL)
- Looked at gels of Linearized Tet BB for GFP and GFP
Sara
- Ran test gel of 08.01.16 GFP PCR:
- 2 uL of Sybr Green, ~30 mL 1% agarose
- 5 uL PCR reaction per piece + 1 uL of 6X Blue Loading Dye
- 6 uL loaded in each well
- 2 NEB 2 kb ladders run per gel, 2uL of 2kb ladder + 6uL 6X Blue Loading Dye
- Dpn1 digest of 08.01.16 GFP PCR with Shu
- 0.5 uL Dpn1 in each 50 uL tube
- Incubated for 2 hours at 37°C
- Lab notebook labelling and organizing
- Read up on golden gate procedures
- Emailed Patrick
Shu
- PCR of GFP again with Sara
- Ran a final gel and gel extracted the GFPs
- Ran a final gel on the digested product, 10uL loading dye in each product, 25uL in each well
Tasfia
- Ran gel of 8.1 PCR Tet Backbone Linearization for GFP/mCherry
- Gel extraction of Tet Lrz for GFP
- Lane 1: 219 mg gel excised, dissolved in 657 μL GEX buffer
- Lane 2: 300 mg gel excised, dissolved in 900 μL GEX
- Lane 3: 271 mg gel excised, dissolved in 813 μL GEX
- Lane 4: 219 mg gel excised, dissolved in 657 μL GEX
- Lane 4: 219 mg gel excised, dissolved in 657 μL GEX
- Used EPOCH’s iGEM-modified protocol for purification
- Let product sit for 1-2 minutes between washes
- Washed with WS buffer twice
- Let product sit for 5 minutes for more optimal elution
- Used nuclease-free water instead of elution buffer
- Results: (4 tubes; data has been averaged from four NanoDrop runs per tube)
Tube
Concentration (ng/uL)
260/280
260/230
1
36.8
1.86
1.12
2
37.3
1.92
1.51
3
27.4
1.90
1.34
4
25.0
1.94
0.65
Tyler
- Ligation of Linearized Tet backbone
- 15.8µL water
- 1.2 µL of backbone
- 2µL of ligase buffer
- 1µL of ligase
- 15 minute wait 2:38-2:53
- Transformation of Gibson Product with Paul
- Looked into dsba SS, designed part
- Began designing split Cas9 primers for gibson assembly (dimerized Cas9)
- Gel on Tet linearization with Thush
- PCR of the Tet Backbone Linearization for GFP/mCherry with Michelle
- Reviewed SS cas9 parts
Michelle
- mCherry Gel Extractions
- mCherry 1 gel parts:
- 0.3712 g, dissolved in 1.1140 mL of GEX buffer
- 0.3501 g, dissolved in 1.0501 mL of GEX buffer
- mCherry 2 gel parts:
- 0.3139 g, dissolved in 0.9417 mL of GEX buffer
- 0.2385 g, dissolved in 0.7155 mL of GEX buffer
- 0.3169 g, dissolved in 0.9507 mL of GEX buffer
- mCherry 3 gel parts:
- 0.3782 g, dissolved in 1.1346 mL of GEX buffer
- 0.3702 g, dissolved in 1.1106 mL of GEX buffer
- 0.3138 g, dissolved in 0.9414 mL of GEX buffer
- mCherry 4 gel parts:
- 0.2850 g, dissolved in 0.855 mL of GEX buffer
- 0.3295 g, dissolved in 0.9885 mL of GEX buffer
- mCherry 5 gel parts:
- 0.3405 g, dissolved in 1.0215 mL of GEX buffer
- 0.3521 g, dissolved in 1.0563 mL of GEX buffer
- 0.3257 g, dissolved in 0.9771 mL of GEX buffer
- Used Epoch’s kit w/ team procedure modifications:
- Washed with WS twice
- Let stand for each wash for 1 minute
- Let stand before spinning for elution for 5 minutes
- Eluted in 20 μL of n.f. water instead of elution buffer
- mCherry 1 gel parts:
- Autoclaved 1 mL tips
- Autoclaved 250 mL of LB for Cam plates
- 0.25 mL of 1000X (35 mg/mL) Cam
- 1 sleeve poured
- HTML & CSS for the website
Paul
- Looked into DsbA as a SS for Cas9
- Transformed Gibson product:
- 50µL competent cells, 5 µL of Gibson product, 1µL of positive control (psb1c3-mrfp), 1µL of negative control (religation of tet backbone)
- -waited ~30 minutes
- 45 second heat shock
- 450µL of SOC added to each tube
- Incubated for an hour
- Plated 20 and 200µL plates for each sample
- Controls:
- Neg: Tet linearized BB+ligase 15 min (used 41 ng/uL BB)
- Pos: pSB1C3_J04450 (Transformation Efficiency Test kit: 50 pg/uL)
- Looked at gels of Linearized Tet BB for GFP and GFP
Sara
- Ran test gel of 08.01.16 GFP PCR:
- 2 uL of Sybr Green, ~30 mL 1% agarose
- 5 uL PCR reaction per piece + 1 uL of 6X Blue Loading Dye
- 6 uL loaded in each well
- 2 NEB 2 kb ladders run per gel, 2uL of 2kb ladder + 6uL 6X Blue Loading Dye
- Dpn1 digest of 08.01.16 GFP PCR with Shu
- 0.5 uL Dpn1 in each 50 uL tube
- Incubated for 2 hours at 37°C
- Lab notebook labelling and organizing
- Read up on golden gate procedures
- Emailed Patrick
Shu
- PCR of GFP again with Sara
- Ran a final gel and gel extracted the GFPs
- Ran a final gel on the digested product, 10uL loading dye in each product, 25uL in each well
Tasfia
- Ran gel of 8.1 PCR Tet Backbone Linearization for GFP/mCherry
- Gel extraction of Tet Lrz for GFP
- Lane 1: 219 mg gel excised, dissolved in 657 μL GEX buffer
- Lane 2: 300 mg gel excised, dissolved in 900 μL GEX
- Lane 3: 271 mg gel excised, dissolved in 813 μL GEX
- Lane 4: 219 mg gel excised, dissolved in 657 μL GEX
- Lane 4: 219 mg gel excised, dissolved in 657 μL GEX
- Used EPOCH’s iGEM-modified protocol for purification
- Let product sit for 1-2 minutes between washes
- Washed with WS buffer twice
- Let product sit for 5 minutes for more optimal elution
- Used nuclease-free water instead of elution buffer
- Results: (4 tubes; data has been averaged from four NanoDrop runs per tube)
Tube Concentration (ng/uL) 260/280 260/230 1 36.8 1.86 1.12 2 37.3 1.92 1.51 3 27.4 1.90 1.34 4 25.0 1.94 0.65
Tyler
- Ligation of Linearized Tet backbone
- 15.8µL water
- 1.2 µL of backbone
- 2µL of ligase buffer
- 1µL of ligase
- 15 minute wait 2:38-2:53
- Transformation of Gibson Product with Paul
- Looked into dsba SS, designed part
- Began designing split Cas9 primers for gibson assembly (dimerized Cas9)
- Gel on Tet linearization with Thush
- PCR of the Tet Backbone Linearization for GFP/mCherry with Michelle
- Reviewed SS cas9 parts