Team:Stockholm/3 August 2010

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(Difference between revisions)
(→Andreas)
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#*25 Cm
#*25 Cm
#Incubation ON in 37 °C
#Incubation ON in 37 °C
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==Mimmi==
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=== MITF ===
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==== - restriction enzyme ====
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 +
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*the PCR products (from 2010-08-02) are treated with the restriction enzyme AgeI
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**the mutated MITF should show two bands: ~1300bp and ~10bp (which you dont see)
 +
**the control (non-mutated) MITF should show three bands: ~1060bp, ~200bp and ~10bp (which you don't see)
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 +
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{|
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! Mix
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| (µl)
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|-
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| DNA
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| 15
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|-
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| sH<sub>2</sub>O
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| 2
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|-
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| 10X buffer
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| 2
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|-
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| AgeI
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| 1
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|-
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| align="right" | tot
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| 20
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|}
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*Mix and spinn down breafly
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*Incubate 2h (1-16h)
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==== - Gel ====
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*0.5% agarose gel
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*(Melted in our bath, redoing the gel in another bath...

Revision as of 20:06, 10 August 2010

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Contents

Andreas

Plasmid prep.

From 2/8 ON cultures

Elution volume: 70 μl.

DNA concentration
Sample Conc. [ng/μl] A260/A280
pSB1A3 1 87.00 1.98
pSB1A3 2 120.3 1.92
pSB1A3 3 145.7 1.94
pSB1A3 4 58.72 2.01
pSB1C3 1 188.9 1.97
pSB1C3 2 101.2 2.00
pSB1C3 3 201.2 1.93
pSB1C3 4 147.7 1.92

Cloning

To remove unwanted insertion of a C nucleotide located at the PstI site in the suffix, caused by cloning in the pEX vector, yCCS, SOD and IgG protease will be transferred to a new vector by digestion with SpeI.

Digestion

  • IgG protease (on pSB1A3)
  • yCCS A (on pSB1C3)
  • yCCS B (on pSB1C3)
  • SOD (on pSB1C3)
  • pSB1C3 2
  • pSB1A3 2
Digestion tubes
[μl] IgGp
[500] 		SOD
[120.6] 		yCCS A
[94.8] 		yCCS B
[85.8] 		pSB1A3
[120.3] 		pSB1C3
[101.2]
10X FastDigest buffer 5 5 5 5 5 5
dH2O 39 26 22 20 26 23
DNA (2 μg) 4 17 21 23 17 20
FD SpeI† 0.5 0.5 0.5 0.5 0.5 0.5
FD EcoRI 1 1 1 1 1 1
  50 50 50 50 50 50

† Only 0.5 μl of FD SpeI to save enzyme, since it is very expensive.

  1. Gentle mixing
  2. Incubation in 37 °C, 15 min
  3. Tubes returned to ice
  4. Promptly proceeded to ligation, without enzyme inactivation or DNA purification.

Ligation

  • pSB1C3 + IgG prot.
  • pSB1A3 + yCCS A
  • pSB1A3 + yCCS B
  • pSB1A3 + SOD
Ligation tubes (vector:insert 1:5)
[μl] IgG + pSB1C3 SOD + pSB1A3 yCCS A + pSB1A3 yCCS B + pSB1A3
Vector DNA [100 ng] 2.5 2.5 2.5 2.5
Insert DNA [500 ng] 12.5 12.5 12.5 12.5
5X Rapid Ligation buffer 4 4 4 4
T4 DNA ligase 1 1 1 1
  20 20 20 20
  1. Gently mixing
  2. Incubation 22 °C, 10 min
  3. Collection by 5 sec centrifugation
  4. Tubes returned to ice

Quick transformation

  1. 100 μl Top10 cells thawed
  2. 2 μl ligation mixture added to cells. Cells kept on ice ≈5 min
  3. Heat-shock in 42 °C, 30 sec
  4. Cells plated on LB agar with relevant antibiotics
    • 100 Amp
    • 25 Cm
  5. Incubation ON in 37 °C



Mimmi

MITF

- restriction enzyme

  • the PCR products (from 2010-08-02) are treated with the restriction enzyme AgeI
    • the mutated MITF should show two bands: ~1300bp and ~10bp (which you dont see)
    • the control (non-mutated) MITF should show three bands: ~1060bp, ~200bp and ~10bp (which you don't see)


Mix (µl)
DNA 15
sH2O 2
10X buffer 2
AgeI 1
tot 20


  • Mix and spinn down breafly
  • Incubate 2h (1-16h)


- Gel

  • 0.5% agarose gel
  • (Melted in our bath, redoing the gel in another bath...
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