Template:Team:UC Berkeley/Notebook/AL notes

From 2008.igem.org

(Difference between revisions)
(→Aronlau 22:08, 11 June 2008 (UTC))
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==[[User:Aronlau|Aronlau]] 18:11, 13 June 2008 (UTC)==
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6/12/2008
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A.  Use Clonewells to isolate DNA samples.  Because sample B5 (K112312) may have been contaminated, PCR for b5 was done again.
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B.  Perform digestion using the follow procedure.
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::1. Make a solution of 5 ul NEB2, 15 ul DNA, 1 ul EcoRI, 1 ul BamHI, 28 ul water
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::2. Incubate for an hour
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C.  Clean up digestion with zymo columns.
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::1.  Add 200ul ADB buffer to each of the digestion samples and pipette into zymo columns.  Spin at 15s at full speed.
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::2.  Add 200ul wash buffer.  Spin for 15s at full speed.
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::3.  Repeat step 2.  Spin for an additional 90s at full speed.
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::4.  Add 7ul of water to tube and spin for 30s at full speed.
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D.  Ligation
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::1.  Mastermix: 6.5ul water, 1ul ligation buffer, 0.5 T4 DNA ligase, 1ul pBca1256.  Aliquot 9ul.
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::2.  Add 1ul of insert.
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::3.  Cover with foil and incubate for 30 min at room temperature.
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E.  Transformation (always keep on ice)
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::1.  220ul competent cell in one tube, thaw on ice.
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::2. Add 30ul KCM and 20 ul water (both cold).
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::3. Invert ~2x to mix.
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::4.  Aliquot 45ul cell into 10 ul of ligation rxn.  (swirl and pipette up and down once)
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::5.  Foil and Incubate 10 min on ice.
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::6.  Heat shock 90s at 42 C.
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::7.  Add 50 ul of LB.
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::8.  Incubate 1 hr at 37 C.
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::9.  Plate
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==[[User:Aronlau|Aronlau]] 22:08, 11 June 2008 (UTC)==
==[[User:Aronlau|Aronlau]] 22:08, 11 June 2008 (UTC)==
6/9/2008
6/9/2008
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::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
::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
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==[[User:Aronlau|Aronlau]] 22:00, 6 June 2008 (UTC)==
==[[User:Aronlau|Aronlau]] 22:00, 6 June 2008 (UTC)==

Revision as of 18:11, 13 June 2008

Aronlau 18:11, 13 June 2008 (UTC)

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.


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

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