Team:Stockholm/11 September 2010

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(Difference between revisions)
(→Andreas)
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====Gel verification====
====Gel verification====
 +
[[image:ColPCR_RBS34_11sep.png|200px|thumb|right|'''Colony PCR gel verification of RBS BBa_B0034 clones.'''<br />4 &mu;l &lambda;; 5 &mu;l sample.<br />50 bp &lambda; = GeneRuler 50 bp DNA ladder; 1 kb &lambda; = O'GeneRuler 1 kb DNA ladder.]]
1.5 % agarose, 90 V
1.5 % agarose, 90 V
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*RBS 34: 250 bp
*RBS 34: 250 bp
*PC: 1385 bp
*PC: 1385 bp
 +
 +
'''Results'''<br />
 +
Relevant bands for clones 1-3. Weak band for clone 4. Selected clone 2 for plasmid prep.
 +
 +
====ON cultures====
 +
Set double ON (until 13/9) cultures for RBS 34.
 +
*5 ml LB + Amp 100
 +
*37 &deg;C, 80 rpm
 +
**pSB1A2.RBS (BBa_B0034)
 +
 +
===Extraction of RBS BioBrick (BBa_B0030)===
 +
 +
====Plasmid prep====
 +
''From 10/9 ON culture''
 +
*Elution volume: 50 &mu;l
 +
*pSB1A2.RBS 30
 +
 +
Stored in -20 &deg;C for later DNA conc. measurement.
 +
 +
====Glycerol stock====
 +
*pSB1A2.BBa_B0030 (RBS) 2010-09-11
 +
**Abbr.: pA.RBS 30
 +
 +
===Cloning and site-directed mutagenesis of MITF===
 +
''From Mimmi's 10/9 ON plate''
 +
 +
Ran a PCR on four colonies from Mimmi's 10/9 transformation plate. Used original "pRc/CMV MITF -M" plasmid as control.
 +
*m-MITF 1-4 11/9 (m-M 1-4)
 +
*MITF plasmid (M)
 +
 +
'''PCR tubes'''<br />
 +
Standard colony PCR tubes (illustra Ready-to-Go PCR beads)
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 +
{|border="1" cellpadding="1" cellspacing="0"
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!colspan="3"|PCR settings
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|-
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|width="50" align="center"|95 &deg;C
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|align="center" colspan="2"|10:00
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|-
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|align="center"|95 &deg;C
 +
|align="center" width="50"|0:30
 +
|rowspan="3"|x5
 +
|-
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|align="center"|55 &deg;C
 +
|align="center"|0:30
 +
|-
 +
|align="center"|72 &deg;C
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|align="center"|1:40
 +
|-
 +
|align="center"|95 &deg;C
 +
|align="center"|0:30
 +
|rowspan="2"|x25
 +
|-
 +
|align="center"|72 &deg;C
 +
|align="center"|2:10
 +
|-
 +
|align="center"|72 &deg;C
 +
|align="center" colspan="2"|10:00
 +
|}
 +
 +
PCR samples stored in 20 &deg;C.

Revision as of 17:56, 13 September 2010

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Contents

Andreas

Preparation of Top10 chemically competent cells

Picked 8 single colonies from the 10/9 LB plate with pipette tips. Colonies were restreaked onto an LB agar plate with Amp 100 to verify that they were not contaminated, and the pipette tips were then used to inoculate cultures of 5 ml LB. Cultures grown for 40 h in 30 °C w/o rotary shaking.

Extraction of RBS BioBrick (BBa_B0034)

Colony PCR

Picked 4 colonies (RBS 1-4) from the 10/9 ON plate and ran a colony PCR.

  • Positive control (PC): pSB1C3.RFP
  • Standard colony PCR protocol
    • Elongation: 0:45

Gel verification

Colony PCR gel verification of RBS BBa_B0034 clones.
4 μl λ; 5 μl sample.
50 bp λ = GeneRuler 50 bp DNA ladder; 1 kb λ = O'GeneRuler 1 kb DNA ladder.

1.5 % agarose, 90 V

Expected bands

  • RBS 34: 250 bp
  • PC: 1385 bp

Results
Relevant bands for clones 1-3. Weak band for clone 4. Selected clone 2 for plasmid prep.

ON cultures

Set double ON (until 13/9) cultures for RBS 34.

  • 5 ml LB + Amp 100
  • 37 °C, 80 rpm
    • pSB1A2.RBS (BBa_B0034)

Extraction of RBS BioBrick (BBa_B0030)

Plasmid prep

From 10/9 ON culture

  • Elution volume: 50 μl
  • pSB1A2.RBS 30

Stored in -20 °C for later DNA conc. measurement.

Glycerol stock

  • pSB1A2.BBa_B0030 (RBS) 2010-09-11
    • Abbr.: pA.RBS 30

Cloning and site-directed mutagenesis of MITF

From Mimmi's 10/9 ON plate

Ran a PCR on four colonies from Mimmi's 10/9 transformation plate. Used original "pRc/CMV MITF -M" plasmid as control.

  • m-MITF 1-4 11/9 (m-M 1-4)
  • MITF plasmid (M)

PCR tubes
Standard colony PCR tubes (illustra Ready-to-Go PCR beads)

PCR settings
95 °C 10:00
95 °C 0:30 x5
55 °C 0:30
72 °C 1:40
95 °C 0:30 x25
72 °C 2:10
72 °C 10:00

PCR samples stored in 20 °C.

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