dspB Track
PCR off gDNA for more PCR products
PCR Master mix
Reagent | 1x rxn volume (uL) | Master Mix |
5x rxn buffer | 5 | x7.5 | 37.5 |
10mM dNTP | 1 | x7.5 | 7.5 |
sdH2O | 6.65 | x7.5 | 49.875 |
Phusion polymerase | 0.1 | x7.5 | .75 |
MgCl2 | 2 | x7.5 | 15 |
DMSO - 5% | 1.25 | x7.5 | 9.375 |
Total | 21 | | 157.5 |
PCR Tubes
Tube | Content |
A1,A2,A3 | dspB his |
B1,B2,B3 | dspB no-his |
W (H2O control) |
- To A1,A2,A3: add 2uL of up-his & dw primers each
- To B1,B2,B3: add 2uL of up & dw primers each
PCR Cycles:
- 98C @ 3min
- Cycle 27x:
- 98C @ 10 sec
- 72C @ 30 sec
- 72C @ 40 sec
- 72C @ 10 min
- 10C @ hold
Start: 0907
End: 1010
Gel verification colony PCR products
- Protocol: gel verification protocol in Protocol (SOP)
- Changes: 1% agarose gel
- Machine conditions: 0.5x TBE buffer, 100V, 45min
Gel orientation:
Gel orientation
A1 | A2 | A3 | 100bp ladder | B1 | B2 | B3 | W (control) |
Results:
O/N cultures
- 2 tubes (3T1, 4T3) taken out at 0950
Miniprep
- Miniprepped 3T1, 4T3
- Protocol: in SOP binder (Alkaline lysis)
Restriction Digest on miniprepped DNA
- See RD protocol (BioBricks)
Gel Verification on mini-prepped RD
- Protocol: gel verification protocol in Protocol (SOP)
- Changes: 1% agarose gel
- Machine conditions: 0.5x TBE buffer,
Gel orientation:
Gel orientation
3T1 | 100bp ladder | 4T3 | chlor |
Results:
Plan: RD + ligation + transformation
Restriction Digest
Protocol: Biobrick Protocol
Restriction Digest Supermix
REAGENTS | 1 RXN VOLUME (uL) | Vicki's SUPERMIX |
Buffer 2 | 5 | x13 | 65 |
BSA | 0.5 | x13 | 6.5 |
ddH2O | 37 | x13 | 481 |
Total | 42.5 | | 552.5 |
- DNA: add 5uL of backbone + 2uL of insert each
- Enzymes: EcoRI and SpeI (1uL each to each tube)
Restriction Digest Supermix
REAGENTS | 1 RXN VOLUME (uL) | Marianne's SUPERMIX |
Buffer 2 | 5 | x33 | 165 |
BSA | 0.5 | x33 | 16.5 |
ddH2O | 37 | x33 | 1221 |
Total | 42.5 | | 1402.5 |
- DNA: add 5uL of backbone + 2uL of insert each
- Enzymes: EcoRI and SpeI (1uL each to each tube)
- RD Tubes:
- AA, TA, CA, AB, TB, CB, 1-16 all contain insert dspB His
- AC, TC, CC< AD, TD, CD, 17-32 all contain insert dspB no-His
Ligation
Calculations
[math]\displaystyle{ ratio \times \frac{insert \rm length}{vector \rm length} \times vector \rm mass = insert \rm mass (ng) }[/math]
[math]\displaystyle{ 3 \times \frac{1200}{2400} \times = ng }[/math]
[math]\displaystyle{ 1uL \rm vector\times\frac{ng \rm vector}{1uL vector}\times\frac{ng \rm insert}{ng \rm vector}\times\frac{uL \rm insert}{x ng \rm insert} = }[/math]
- Where x = concentration of insert
Vicki: ligation - 6:1 ratio using
- amp backbone
- amp+tet backbone
- chlor backbone
Calculations
Tube | Amount of vector(uL) | Amount of insert (uL) |
A | 3 | 2.559 |
B | 3 | 2.808 |
C | 3 | 2.820 |
D | 3 | 3.042 |
Marianne:
Ligation - chlor backbone using ratios:
- 2:1,3:1,4:1,6:1,7:1,8:1,9:1,10:1
Calculations for ratio 2:1
Tube | Amount of vector(uL) | Amount of insert (uL) |
A2 | 3 | 0.852 |
B2 | 3 | 0.936 |
C2 | 3 | 0.941 |
D2 | 3 | 1.014 |
Calculations for ratio 3:1
Tube | Amount of vector(uL) | Amount of insert (uL) |
A3 | 3 | 1.2801 |
B3 | 3 | 1.4046 |
C3 | 3 | 1.4106 |
D3 | 3 | 1.5216 |
Calculations for ratio 4:1
Tube | Amount of vector(uL) | Amount of insert (uL) |
A4 | 3 | 1.707 |
B4 | 3 | 1.872 |
C4 | 3 | 1.881 |
D4 | 3 | 2.028 |
Calculations for ratio 6:1
Tube | Amount of vector(uL) | Amount of insert (uL) |
A6 | 3 | 2.559 |
B6 | 3 | 2.808 |
C6 | 3 | 2.820 |
D6 | 3 | 3.042 |
Calculations for ratio 7:1
Tube | Amount of vector(uL) | Amount of insert (uL) |
A7 | 3 | 2.988 |
B7 | 3 | 3.279 |
C7 | 3 | 3.291 |
D7 | 3 | 3.552 |
Calculations for ratio 8:1
Tube | Amount of vector(uL) | Amount of insert (uL) |
A8 | 3 | 3.414 |
B8 | 3 | 3.747 |
C8 | 3 | 3.762 |
D8 | 3 | 4.059 |
Calculations for ratio 9:1
Tube | Amount of vector(uL) | Amount of insert (uL) |
A9 | 3 | 3.840 |
B9 | 3 | 4.215 |
C9 | 3 | 4.233 |
D9 | 3 | 4.566 |
Calculations for ratio 10:1
Tube | Amount of vector(uL) | Amount of insert (uL) |
A10 | 3 | 4.266 |
B10 | 3 | 4.583 |
C10 | 3 | 4.701 |
D10 | 3 | 5.073 |
Transformation
- Protocol: Transformation protocol from SOP
- Changes: 10uL of ligation mix instead of 1uL; 1 hour incubation instead of 2 hours
Biofilm Track
Eric F. + Melody
8325-4 Biofilm Growth
Streaked a TSA plate with 8325-4 TSB O/N culture and put in incubator at 37°C
RN4220 Biofilm Growth
Completed Day 3 of the protocol for both plates
- Test #1 - control E5 showed growth
- Test #2 - controls B2, B11, C5, E7 and G2 showed growth
- We decided to continue the experiment as a wet run (due to the contamination the results are meaningless)
- Next time we will perform the inoculation step in the bio safety cabinet
- The 60°C air dry step was completed using a heat block
- For the overnight step, we placed the plates in the bio safety cabinet
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