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Team:Illinois/Antibody GPCR Fusion Notebook
From 2008.igem.org
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|Buffer G | |Buffer G | ||
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|x4 | |x4 | ||
|50uL | |50uL | ||
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| + | |Forward Primer | ||
| + | |0.5uL | ||
| + | |x4 | ||
| + | |2uL | ||
| + | |- | ||
| + | |Reverse Primer | ||
| + | |0.5uL | ||
| + | |x4 | ||
| + | |2uL | ||
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|template | |template | ||
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* Prepped 3 overnight cultures | * Prepped 3 overnight cultures | ||
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== 28th August == | == 28th August == | ||
Revision as of 02:38, 10 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 (FILL IN PCR TABLE)
| Buffer G | 12.5uL | x4 | 50uL | |||||
| Forward Primer | 0.5uL | x4 | 2uL | |||||
| Reverse Primer | 0.5uL | x4 | 2uL | |||||
| template | 0.5ul of both heavy and light chains | |||||||
| primers | 1ul of appropriate forward and reverse primer | |||||||
| MgCl2 | 0ul | 0ul | 3ul | 3ul | ||||
| master mix | 20ul | |||||||
| H20 | to 50ul total volume | |||||||
PCR program: 1. 94 degrees 5 min 2. 94 degrees 1 min 3. 50 degrees 1 min 4. 72 degrees 1 min 5. GOTO 2. 29 cycles 6. HOLD at 4 degrees
Products run on a 1.5% agarose gel, no products of 700-800bp.
- 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
- FILL IN PCR TABLE
3rd September
- Ran gel
- Ladder lane 7
- Sample 7 spilled
- 1% agarose
- Too high
- 120V
- Too low
- 50 minutes
8th September
- FILL IN PCR TABLE
- 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
- FILL IN PCR TABLE
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
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