Team:Stockholm/30 August 2010

From 2010.igem.org

(Difference between revisions)
(→Andreas)
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*2 μl ligation mix
*2 μl ligation mix
*Cm 25
*Cm 25
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== Mimmi ==
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=== MITF-M ===
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==== Colony PCR ====
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 +
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*Made by Andreas --> ~280bp bands, empty vector? should not be possible...
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*Try again, more colonies
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{|
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! Mix
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| (µl)
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| X8
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| rowspan="8" width="150" |
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! Primers
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| rowspan="8" width="150" |
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! colspan="2" | Conditions
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| rowspan="3" |
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|-
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| sH<sub>2</sub>O
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| 22.5
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| 180
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| pSB1_VF2
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! time
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! &deg;C
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|-
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| F primer
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| 1
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| 8
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| pSB1_VR
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| 2m
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| 94
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|-
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| R primer
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| 1
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| 8
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| rowspan="5" |
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| 30s
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| 94
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| )
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|-
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| DNA
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| 0.5
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| 8x0.5
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| 30s
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| 50
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| > 30 cycles
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|-
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| align="right" | tot
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| 25µl
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|
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| 2m40s
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| 72
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| )
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|-
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| rowspan="2" colspan="3" |
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| 10m
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| 72
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| rowspan="2" |
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|-
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| oo
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| 10
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|}
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==== Gel ====
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{|
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! well
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! sample
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|-
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| 1
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| ladder
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|-
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| 2
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| pSB1C3.MITF-M 1
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|-
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| 3
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| pSB1C3.MITF-M 2
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|-
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| 4
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| pSB1C3.MITF-M 3
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|-
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| 5
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| pSB1C3.MITF-M 4
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|-
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| 6
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| pSB1C3.MITF-M 5
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|-
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| 7
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| pSB1C3.MITF-M 6
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|-
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| 8
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| pSB1C3.MITF-M 7
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|-
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| 9
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| blank
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|}
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 +
 +
*No product, trying another 7 colonies over night...

Revision as of 15:32, 31 August 2010

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Contents

Andreas

Cloning of SOD into pMA.His

Transformation results

From 28 28/8 Good colony yield. Four colonies (SH1-SH4) picked for colony PCR.

Colony PCR

  • SH1-SH4: pMA.SOD⋅His
  • PC: Positive control; pMA.His
  • NC: Negative control; blank

Procedures according to standard colony PCR protocol. Elongation time 1:00.

Gel verification

Gel verification of SOD cloning into pMA.His.
3 μl λ, 4 μl sample.
λ=O'GeneRuler 1 kb DNA ladder.

1 % agarose, 100 V

Expected bands:

  • pMA.SOD⋅His: 831 bp
  • pMA.His: 348 bp

Results
Well corresponding bands indicating successful insertion of SOD into the vector.

ON cultures

SH1 and SH2 selected for plasmid prep and sequencing. Set 5 ml LB + 100 Amp ON cultures. 37 °C, 225 rpm.

N-CPP extraction

Gel extraction

From 28/8 samples Purification using the E.Z.N.A. Gel Extraction kit. Elution in 30 μl dH2O; double elution.

† Phenolate contaminated sample? High absorbtion at 230 nm [http://en.wikipedia.org/wiki/Nucleic_acids_analysis#Other_common_contaminants]
DNA concentrations
Sample Conc. [ng/μl] A260/A280
Tra10 13.56 1.86
TAT 1.736 1.20
LMWP † 2.523 2.69

Gel verification

Gel verification of extracted N-CPPs.
3 μl λ; 2 μl sample.
λ=GeneRuler 50 bp DNA ladder

Ran a gel to verify that the presence and size of our extracted DNA fragments.

1 % agarose, 100 V

Results
Weak band for Tra10, no bands for TAT and LMWP. Proceeded to cloning anyway.

Cloning of N-CPPs into pSB1C3

A last-minute decision was made to also make a bulk cloning of all three N-CPPs by digesting directly from the N-CPP cluster vector.

Digestion

[N-CPP plasmid]=672 ng/μl (28/8)

[ng/μl] Tra10 TAT LMWP N-CPP
10X FD buffer 3 3 3 3
dH2O 3 3 8 23
FD XbaI 0.5 0.5 0.5 0.5
FD AgeI 0.5 0.5 0.5 0.5
DNA 23 23 18 3
  30 30 30 30

Incubation: 37 °C, 30 min
Inactivation: 80 °C, 10 min

Dephosphorylation

Treated the N-CPP sample with FastAP alkaline phosphatase to prevent multiple insertions (3, 5, etc...) into target vector.

  • 3 μl FastAP
    • Incubation: 37 °C, 10 min
  • Inactivation at 75 °C, 5 min

Ligation

[Dig. pSB1X3 X+A EXTR]=13.72 ng/μl (digested and extracted vector from 9/8) [Dig. N-CPP X+A]=60 ng/μl

[ng/μl] N-CPP Tra10 TAT LMWP
Vector DNA 3 3 3 3
Insert DNA 9 12 12 12
5X Rapid Lig. buf. 4 4 4 4
dH2O 3 0 0 0
T4 DNA Ligase 1 1 1 1
  20 20 20 20

Transformation

Standard transformation protocol.

  • 2 μl ligation mix
  • Cm 25



Mimmi

MITF-M

Colony PCR

  • Made by Andreas --> ~280bp bands, empty vector? should not be possible...
  • Try again, more colonies


Mix (µl) X8 Primers Conditions
sH2O 22.5 180 pSB1_VF2 time °C
F primer 1 8 pSB1_VR 2m 94
R primer 1 8 30s 94 )
DNA 0.5 8x0.5 30s 50 > 30 cycles
tot 25µl 2m40s 72 )
10m 72
oo 10


Gel

well sample
1 ladder
2 pSB1C3.MITF-M 1
3 pSB1C3.MITF-M 2
4 pSB1C3.MITF-M 3
5 pSB1C3.MITF-M 4
6 pSB1C3.MITF-M 5
7 pSB1C3.MITF-M 6
8 pSB1C3.MITF-M 7
9 blank


  • No product, trying another 7 colonies over night...
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