Glasgow/Wetlab/Week3

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== Week 3 ==
== Week 3 ==
=== Tuesday 17th July 2007 ===
=== Tuesday 17th July 2007 ===
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#Restriction Digests (see [[Glasgow/Wetlab/Protocols#Protocol 7: Restriction Digests|Protocol 7]]):
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#Restriction Digests of transformed BioBricks (see [[Glasgow/Wetlab/Protocols#Protocol 7: Restriction Digests|Protocol 7]]):
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#*BBa_J23119 (strong constitutive promoter) pSB1A2.  1 x NheI, 1 x PvuI.  680bp, 1430bp
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{| border="1" cellspacing="0" cellpadding="5" align="center"
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#*BBa_R0062 (HSL and luxR) pBB1A2.  1 x EcoRI, 1 x PvuI. 1460bp, 660bp
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!Label
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#*BBa_J04500 (IPTG inducer and RBS) pSB1AK3.  1 x PvuII, 2 x PvuI. 2250bp, 1030bp, 730bp
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!BioBrick
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!Plasmid
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!Description
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!Enzymes
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!Expected Sizes
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|-
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|3/19A
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|BBa_J23119  
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|pSB1A2
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|strong constitutive promoter
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|NheI, PvuI  
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|680bp, 1430bp
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|-
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|1/9G
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|BBa_R0062  
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|pBB1A2
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|HSL and luxR
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|EcoRI, PvuI
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|1460bp, 660bp
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|-
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|1/16P
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|BBa_J04500
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|pSB1AK3
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|IPTG inducer and RBS
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|PvuII, PvuI
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|2250bp, 1030bp, 730bp
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|
#*BBa_p1010 (death gene) pSB3K3. 1 x BamHI, 2 x XhoI. 190bp, 2390bp,840bp.  
#*BBa_p1010 (death gene) pSB3K3. 1 x BamHI, 2 x XhoI. 190bp, 2390bp,840bp.  
#*BBa_E0040 (GFP no promoter) pSB1A2. 1 x Hime II, 1 x PvuI. 1630bp, 1170bp.
#*BBa_E0040 (GFP no promoter) pSB1A2. 1 x Hime II, 1 x PvuI. 1630bp, 1170bp.
#*BBa_I52001 (6D) p5B4K5. 2 x AvaI, 1 x PvuI. 920bp, 1470bp, 2120bp.
#*BBa_I52001 (6D) p5B4K5. 2 x AvaI, 1 x PvuI. 920bp, 1470bp, 2120bp.
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|}
=== Wednesday 18th July 2007 ===
=== Wednesday 18th July 2007 ===

Revision as of 15:28, 17 August 2007

Glasgow Main Page | Back To Wetlab Log

Contents

Week 3

Tuesday 17th July 2007

  1. Restriction Digests of transformed BioBricks (see Protocol 7):
Label BioBrick Plasmid Description Enzymes Expected Sizes
3/19A BBa_J23119 pSB1A2 strong constitutive promoter NheI, PvuI 680bp, 1430bp
1/9G BBa_R0062 pBB1A2 HSL and luxR EcoRI, PvuI 1460bp, 660bp
1/16P BBa_J04500 pSB1AK3 IPTG inducer and RBS PvuII, PvuI 2250bp, 1030bp, 730bp
    • BBa_p1010 (death gene) pSB3K3. 1 x BamHI, 2 x XhoI. 190bp, 2390bp,840bp.
    • BBa_E0040 (GFP no promoter) pSB1A2. 1 x Hime II, 1 x PvuI. 1630bp, 1170bp.
    • BBa_I52001 (6D) p5B4K5. 2 x AvaI, 1 x PvuI. 920bp, 1470bp, 2120bp.

Wednesday 18th July 2007

  1. Last night we diluted primers to 100 pmol/ul by adding the amount of dH2O given on the order sheet, and made working solutions to 10 pmol/ul (2 x 200 ul each - 20 ul stock and 180 ul dH2O).
  2. Maija ran a gel of the restriction digests to check the sizes of the biobricks. These were the maps we made yesterday.
  3. Maia is extracting DNA from 3 samples of ****, which were plated yesterday and grown in liquid media, using MoBio PowersoilTM DNA purification kit (see Protocol 8); only change was to shorten "shaking" time from 10 to 2 minutes.
  4. PCR trial run using Reddymix and Touch 2 done on:
    • pGLTUR: XylR_prefix / XylR_suffix, Pr_prefix / Pr_suffix, Pr_prefix / XylR_suffix, Pu_prefix_EM / Pu_suffix_EM.
    • pQF52 plasmid: DntR_prefix / DntR_suffix
    • DmpR WT/24: DntR_prefix / DntR_suffix
    • Biobricks: R0062, E0040, J04500, J23119, p1010, I52001.
    • See Protocol 9 for PCR.

Thursday 19th July 2007

  1. Maia redid PCR on pGLTUR and pQF52 using Reddymix and a gradient program (See Protocol 9) to minimise the non-specific amplification evident in previous gels. New DntR suffix primer has arrived. Used the following primer sets.
    • XylR prefix and XylR suffix
    • Pr prefix and Pr suffix
    • Pr prefix and XylR suffix
    • Pu prefix and Pu suffix
    • DntR prefix and DntR suffix 2

Friday 20th July 2007

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