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Template:Team:UC Berkeley/Notebook/AL notes
From 2008.igem.org
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A. Received sequencing data. All perfect aside from K112311 with point mutation and K112316 with point mutation in EcoRI (possible poor signal so assumed it is perfect). | A. Received sequencing data. All perfect aside from K112311 with point mutation and K112316 with point mutation in EcoRI (possible poor signal so assumed it is perfect). | ||
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6/23/2008 | 6/23/2008 | ||
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A. Bing minipreped. | A. Bing minipreped. | ||
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6/21/2008 | 6/21/2008 | ||
A. Madhvi picked colonies for K112311, K112313, K112316 and K112319-1 and K112319-2. | A. Madhvi picked colonies for K112311, K112313, K112316 and K112319-1 and K112319-2. | ||
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6/20/2008 | 6/20/2008 | ||
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E. Religated and transformed for K112311, K112313, and K112316. | E. Religated and transformed for K112311, K112313, and K112316. | ||
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6/19/2008 | 6/19/2008 | ||
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B. Perform PCR for K112319 A and B and separated with Clonewell. Started overlap extension. | B. Perform PCR for K112319 A and B and separated with Clonewell. Started overlap extension. | ||
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6/18/2008 | 6/18/2008 | ||
A. Received sequencing data. For K112319, there was a restriction site that was missed when designing oligos. Redesigned oligos. For parts with different sequences or point mutation, picked colonies. | A. Received sequencing data. For K112319, there was a restriction site that was missed when designing oligos. Redesigned oligos. For parts with different sequences or point mutation, picked colonies. | ||
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6/17/2008 | 6/17/2008 | ||
Revision as of 17:52, 24 June 2008
Contents |
Aronlau 17:51, 24 June 2008 (UTC)
6/24/2008
A. Received sequencing data. All perfect aside from K112311 with point mutation and K112316 with point mutation in EcoRI (possible poor signal so assumed it is perfect).
6/23/2008
A. Sent out sample for sequencing
6/22/2008
A. Bing minipreped.
6/21/2008
A. Madhvi picked colonies for K112311, K112313, K112316 and K112319-1 and K112319-2.
6/20/2008
A. Clonewell to isoolate K112319.
B. Digestion and Zymocleanup
C. Ligation
D. Transformation
E. Religated and transformed for K112311, K112313, and K112316.
6/19/2008
A. Bing minipreped the colonies from yesterday and sent out for sequencing.
B. Perform PCR for K112319 A and B and separated with Clonewell. Started overlap extension.
6/18/2008
A. Received sequencing data. For K112319, there was a restriction site that was missed when designing oligos. Redesigned oligos. For parts with different sequences or point mutation, picked colonies.
6/17/2008
A. Bing minipreped and sent out for sequencing.
Aronlau 17:04, 17 June 2008 (UTC)
6/16/2008
A. For colonies with perfect match in sequencing, we made a stock of them on a plate.
- 1. Transformed the plasmids sent in for sequencing into compotent cells.
- a. Mix of 220ul competent cells, 30ul KCM, 50ul water. Aliquot 30ul.
- b. Add mix to 1ul of plasmid.
- c. Incubate on ice for 10 min, heat shock at 42 C for 1.5 min, incubate on ice for 2 min.
- d. Transfer to plate.
- 1. Transformed the plasmids sent in for sequencing into compotent cells.
B. For colonies with point mutation, bad read, or other sequencing, we picked new colonies.
6/15/2008
The DNA sent for sequencing contained a lot of RNA, which affected some of the sequencing. Results for sequencing for pBca1256-K1112300 to pBca1256-K112320
6/14/2008
Bing came in to do a miniprep of the bacteria I picked yesterday using the Agencourt miniprep kit. This kit uses magnetic beads that bind dna to purify dna. The dna was sent out for sequencing analysis.
Aronlau 18:11, 13 June 2008 (UTC)
6/13/2008
A. Waited for colonies to grow larger.
B. Made 2xYT media and aliquoted 1ml.
C. Grew up the E.Coli in a 96 well plate.
6/12/2008
A. Use Clonewells to isolate DNA samples. Because sample B5 (K112312) may have been contaminated, PCR for b5 was done again.
B. Perform digestion using the follow procedure.
- 1. Make a solution of 5 ul NEB2, 15 ul DNA, 1 ul EcoRI, 1 ul BamHI, 28 ul water
- 2. Incubate for an hour
C. Clean up digestion with zymo columns.
- 1. Add 200ul ADB buffer to each of the digestion samples and pipette into zymo columns. Spin at 15s at full speed.
- 2. Add 200ul wash buffer. Spin for 15s at full speed.
- 3. Repeat step 2. Spin for an additional 90s at full speed.
- 4. Add 7ul of water to tube and spin for 30s at full speed.
D. Ligation
- 1. Mastermix: 6.5ul water, 1ul ligation buffer, 0.5 T4 DNA ligase, 1ul pBca1256. Aliquot 9ul.
- 2. Add 1ul of insert.
- 3. Cover with foil and incubate for 30 min at room temperature.
E. Transformation (always keep on ice)
- 1. 220ul competent cell in one tube, thaw on ice.
- 2. Add 30ul KCM and 20 ul water (both cold).
- 3. Invert ~2x to mix.
- 4. Aliquot 45ul cell into 10 ul of ligation rxn. (swirl and pipette up and down once)
- 5. Foil and Incubate 10 min on ice.
- 6. Heat shock 90s at 42 C.
- 7. Add 50 ul of LB.
- 8. Incubate 1 hr at 37 C.
- 9. Plate
Aronlau 22:08, 11 June 2008 (UTC)
6/9/2008
A. Filled out PCR table for parts K112300-K112321 to speed up PCR time when oligos arrive.
6/10/2008
Note: Oligos came and diluted
A. Made the vector pBca1256
- 1. PCR-50 ul
- a. 43 ul water, 1 ul 10mM dNTP, 5 ul 10x Expand Buffer, 1 ul 10uM ca1246F, 1ul 10uM ca1246R, 0.75 ul polymerase, 0.5 ul template (pBca1256)
- 2. Zymo cleanup
- 3. Digest vector using 1ul of EcoRI, BamHI, DpnI and 87 DNA/water solution. Jin ran a gel and said it is fine.
- 1. PCR-50 ul
6/11/2008
A. PCR for parts K112300-K112321-First Try **Refer to construction files for oligos**
- 1. Added 5x more dNTPs, which competes for Mg in buffer with polymerase.
- a. 18.5 ul water, 2.5 ul 10 mM dNTP, 1 ul buffer, 0.5 ul polyermase, 0.5 ul template, 1ul oligo 1, 1 ul oligo 2
B. PCR for parts K112300-K112321-Second Try
- 1. 20.375 ul water, 0.5 ul 10 mM dNTP, 1ul buffer, 0.375 ul polyermase, 0.25 ul template, 1ul oligo 1, 1ul oligo 2
Aronlau 22:00, 6 June 2008 (UTC)
6/2/2008 to 6/5/2008
A. Safety Training
B. Cloning Training-PCR, Purification, Digestion, Miniprep, Transformation, Autoclaving
6/4/2008 to 6/6/2008 - Design Oligos/Make Construction file
A. Oligos al0001 to al0024 for parts K112300-K112321
