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Team:Illinois/Antibody GPCR Fusion Notebook
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Revision as of 02:17, 28 October 2008
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Recipes
- Tris-Cl, 1M
- Dissolve 121g Tris base in 800ml H2O
- Adjust to desired pH with concentrated HCl
- Mix and add H2O to 1 liter
- (Approximately, 20ml HCl for pH 7.4 and 42ml for pH 8.0)
- EDTA, 0.5M (pH 8.0)
- Dissolve 186.1g Na2 EDTA-2H2O in 700ml H20
- Adjust pH to 8.0 with 10M NaOH(~50ml)
- Add H2O to 1 liter
- Breaking buffer - 100ml
- 2ml Triton X-100
- 1ml Sodium dodecyl sulfate (SDS)
- 0.5844g NaCl (100mM)
- 1ml 1M Tris-Cl pH 8.0 (10mM)
- 200uL 0.5M EDTA (1mM)
22nd July
- Yeast obtained from Dr. Zhao
24th July
- Prepared liquid culture for DNA extraction
- Made 1M Tris. Cl pH 8.0
- Made 4M ammonium acetate
22nd August
- Attempted DNA extraction
- Result: Failed
- Obtained more yeast from Dr. Zhao
25th August
- Prepared overnight culture for DNA extraction (3:27pm)
26th August
- Attempted DNA extraction
- Prepped overnight culture
27th August
- Performed PCR: PGK Terminator
| Buffer G | 12.5uL | x4 | 50uL | |
| Forward Primer | 0.5uL | x4 | 2uL | |
| Reverse Primer | 0.5uL | x4 | 2uL | |
| H2O | 10.8uL | x4 | 43.2uL | |
| Taq | 0.2uL | x4 | 0.8uL | |
| template | 0.5ul | |||
| Negative control | 3 H2O | |||
- PCR program:
- 4 min 94 degrees
- 25-30x 30s 94 degrees
- 30s Tm primers
- 1 min/KB 72 degrees
- 7 min 72 degrees
- For the gel: 5uL loading dye gel is in cold room
- Prepped 3 overnight cultures
28th August
- Extracted DNA from 4 cultures
- Ran gel of PCR products (1.5%, 200V)
- Result: No bands present
2nd September
- PCR: PGK Terminator
| Mastermix | 10uL | x3 | 30uL |
| Forward Primer | 0.5uL | x3 | 1.5uL |
| Reverse Primer | 0.5uL | x3 | 1.5uL |
| Template | 10uL | x3 | 30uL |
| H2O | 10.8uL | x4 | 43.2uL |
| Negative control | 25uL H2O |
3rd September
- Ran gel
- Ladder lane 7
- Sample 7 spilled
- 1% agarose
- Too high
- 120V
- Too low
- 50 minutes
8th September
- PCR: PGK Terminator
| Mastermix | 10uL | x3 | 30uL |
| Forward Primer | 0.5uL | x3 | 1.5uL |
| Reverse Primer | 0.5uL | x3 | 1.5uL |
| Template | 14uL | x3 | 42uL |
- Prepped 4 overnight cultures
- Yeast dried out again
9th September
- Signs of life in 3 of the cultures
- Wait until tomorrow
- Ran gel on PCR from 8th September
- 150V, 50 minutes
- No sign of DNA
- Ladder from Courtney
10th September
- Ran gel again
- Split culture
- 150V, 50 minutes
- 0.75% gel
- Ladder from Courtney
11th September
- PCR: PGK Terminator
| Mastermix | 10uL | x3 | 30uL |
| Forward Primer | 0.5uL | x3 | 1.5uL |
| Reverse Primer | 0.5uL | x3 | 1.5uL |
| Template | 14uL | x3 | 42uL |
12th September
- Isolated DNA from 8 cultures
- Ran gel
- 1% agarose
- 150V
- 38 mins
- Poor results
15th September
- PCR PGK Promotor
- Finnzymes Phusion High Fidelity DNA Polymerase
- F-530, 20V (2V/uL)
- Finnzymes Phusion High Fidelity DNA Polymerase
- FiLL out PCR TABLE
18th September
- Ran ___? reaction
- Extracted DNA from gel from 8th September (PGK Terminator)
- FILL TABLE
19th September
- Gel of PGK Promotor has no DNA present
23rd September
- PCR: Fus1 Downstream
- Fill PCR TABLE
24th September
- Ran gel of Fus1 Downstream
- Result: No DNA present on gel
25th September
- PCR: Fus1 Upstream
1st October
- PCR: Ste2
8th October
- PCR: PGK Terminator
- Use DNA extracted from gel on 18th September
- Also extracted DNA from gel from 30th September
9th October
- PCR: Ste2
- Template used is product from 1st October
- FIll IN TABLE
- PCR: Fus1 Upstream
- Template used was extracted from 30th September on 8th September
- Fill in PCR TABLE
- Ran Ste2 gel
14th October
- Fus1 PCR
- Template is reaction from 12th October
15th October
- Gel of Fus1 -> 3 streaks -> no ladder
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