Difference between revisions of "Team:Stony Brook/Notebook/Cancer-W9"
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<h4> Digest of BioBricks </h4> | <h4> Digest of BioBricks </h4> | ||
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<h5> J23119 - 225.8ng</h5> | <h5> J23119 - 225.8ng</h5> | ||
Revision as of 18:05, 26 August 2016
Week 9: 8/22 - 8/28
8/22
PCR of new IDT Shipments
AmilCP
| Contents | ul | Q5 MasterMix | 12.5 |
|---|---|
| H2O | 9 |
| 10 uM F Primer | 1.25 |
| 10 uM R Primer | 1.25 |
| Template | 1 |
IDT #1
| Contents | ul | Q5 MasterMix | 12.5 |
|---|---|
| H2O | 6.5 |
| 10 uM F Primer | 2.5 |
| 10 uM R Primer | 2.5 |
| Template | 1 |
IDT #2
| Contents | ul | Q5 MasterMix | 12.5 |
|---|---|
| H2O | 6.5 |
| 10 uM F Primer | 2.5 |
| 10 uM R Primer | 2.5 |
| Template | 1 |
PCR Settings: 25 Cycles
| Phase | Temperature (°C) | Time (sec) |
|---|---|---|
| Initial Denaturation | 98 | 30 |
| Denaturation | 98 | 10 |
| Annealing | 65 | 30 |
| Extension | 72 | 120 |
| Final Extension | 72 | 120 |
Digest of BioBricks
J23119 - 225.8ng
| Content | Experimental ul | Control ul |
|---|---|---|
| Cutsmart | 5 | 5 |
| EcoRI-HF | 0.5 | X |
| SpeI | 0.5 | X |
| DNA | 44.3 | 2.2 |
B0015 - 201.5ng
| Content | Experimental ul | Control ul |
|---|---|---|
| Cutsmart | 5 | 5 |
| XbaI | 0.5 | X |
| PstI | 0.5 | X |
| DNA | 49.6 | 2.5 |
E0040 - 179.3ng
| Content | Experimental ul | Control ul |
|---|---|---|
| Cutsmart | 5 | 5 |
| EcoRI-HF | 0.5 | X |
| SpeI | 0.5 | X |
| DNA | 55.8 | 2.8 |
LB Prep
J23119
- 8ml LB
- 2ml chloramphenicol
K592009
- 8ml LB
- 2ml chloramphenicol
E0040
- 8ml LB
- 2ml Ampicillin
B0015
- 8ml LB
- 2ml chloramphenicol
8/23
PCR of amilCP, #1 and #2 with 67° annealing temperature
- Ran a gel afterwards
Lanes:
- Ladder
- AmilCP
- #1
- #2
Gel of 10ug overnight BB digest
Lanes:
- Ladder
- J23119
- J23119 Control
- B0015
- B0015 Control
- E0040
- E0040 Control
Competent Cells (TSS)
50ml TSS
| Content | Amount |
|---|---|
| PEG3350 | 5g |
| 1M MgCL2 | 1.5 ml | DMSO | 2.5ml |
| LB | Fill to 50ml |
- Tested pH to be ~6.5
New PCR of Toggle #1 and amil CP
- 69°C annealing temperature
- Extension length - 2.5 mins
- Ran for 30 Cycles
Ran a gel
Lanes:
- amil CP
- #4
- Ladder
Gel of Digested BB Parts
Lanes:
- Ladder
- J23119 #1
- J23119 #2
- B0015 #1
- B0015 #2
- E0040 #1
- E0040 #2
Nanodrops of BioBricks and PCR purifications
| Content | Concentration (ng/ul) | 260/280 |
|---|---|---|
| R592009 | 234.3 | 1.92 |
| J23119 | 93.0 | 1.95 |
| B0015 | 229.4 | 2.00 |
| E0040 | 287.5 | 1.95 |
| PiggyBac | 274.0 | 1.89 |
| Toggle #2 | 9.6 | 2.45 |
Digests
- Re-do of the the digest from 8/21
PCR of Amil CP and #1
- 67°C annealing temperature for 15 seconds
- 60 second extension
25ul PCR of Toggle #2
- 68°C annealing temperature
- Primer concentrations at 100-500 ng/ul
Gel of BioBrick Digests
Gel #1
Lanes:
- Ladder
- B0015
- B0015
- B0015 Control
- J12119
- J23119
- J23119 Control
Gel #2
Lanes:
- Ladder
- K592009
- K592009
- K592009
- K592009 Control
- E0040
- E0040
- E0040 Control
PCR of Toggle #2 - 30x
| Contents | ul | Q5 MasterMix | 12.5 |
|---|---|
| H2O | 5.5 |
| 10 uM F Primer | 1.0 |
| 10 uM R Primer | 1.0 |
| Template | 5 |
| Contents | ul | Q5 MasterMix | 12.5 |
|---|---|
| H2O | 3.5 |
| 10 uM F Primer | 2.0 |
| 10 uM R Primer | 2.0 |
| Template | 5 |
Gel
Lanes:
- Ladder
- Toggle #2 - 1ul primer
- Toggle #2 - 2ul primer
Toggle #1 Trials
PCR of Toggle #1
- Ran 6 trials. The first started with 0.5 ul of each of the F and R primers.
- Each trial increased by 0.5 until we got to 2.5ul of primers
- Ran a 0.25ul primer trial
- Used 12.5ul of Q5 MasterMix and 5ul of template
Toggle #1 PCR
Lanes:
- Ladder
- 0.25
- 0.5
- 1.0
- 1.5
- 2.0
- 2.5
8/24
