Team:Stockholm/27 July 2010

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(→Colony PCR on IgG protease in shipping vector)
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{{Stockholm/Top2}}
{{Stockholm/Top2}}
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== Mimmi ==
 +
 +
 +
=== over-expression in pEX ===
 +
::::- '''remaking the comassie-gel'''
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 +
 +
 +
{| border="1" cellspacing="0" cellpadding="2"
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|-
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!scope="col"| '''Mix'''
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!scope="col"| v
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|-
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!scope="row"| LB
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| 4 ml
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|-
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!scope="row"| glycose
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| 40 µl (1%)
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|-
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!scope="row"| Amp
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| 8 µl (0.5%)
 +
|-
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!scope="row"| old culture
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| 40 µl
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|}
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 +
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*Grow in 37C, ~200 rpm ~2h until OD = 0.6
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 +
*Add IPTG (1 µl/ml,1M)in one of the two cultures, the other one is used as a control
 +
 +
*Take sample at 0h, 1h, 2h, 3h
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**pipette 500µl into a 1.5ml tube
 +
**spinn down the sells and remove LB
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**resuspend in 50µl loading dye
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**freeze
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 +
 +
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=== Site-Directed Mutagenesis ===
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 +
==== Primers ====
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:(MW/10)/(OD*33) = dilution factor
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::1000/df = volyme to get 100µM primer
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:::100µM = (MW/10) ng/µl
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::::(MW/10)/125 = df to get 125 ng/µl
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::{| border="1" cellspacing="0" cellpadding="2"
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|-
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!scope="col"| '''primer'''
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!scope="col"| V H2O (µl)
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!scope="col"| V H2O to get 125 ng/µl (µl)
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|-
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!scope="row"| MITF_site1_F
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| 463.88
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| 3µl + 15.4µl
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|-
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!scope="row"| MITF_site1_R
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| 64.77
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| 3µl + 15.3µl
 +
|-
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!scope="row"| MITF_site2_F
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| 144.85
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| 3µl + 21µl
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|-
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!scope="row"| MITF_site2_R
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| 141.77
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| 3µl + 21.6µl
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|-
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!scope="col"| .
 +
!scope="col"| concentration
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!scope="col"| .
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|-
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!scope="row"| yCCS_F
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| 1085.7 ng/µl
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| 3µl + 23µl
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|-
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!scope="row"| yCCS_R
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| 996.5 ng/µl
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| 3µl + 20.9µl
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|}
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 +
==== reaction ====
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{| border="1" cellspacing="0" cellpadding="2"
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|-
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!scope="col"| '''Mix'''
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!scope="col"| (µl)
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!scope="col"| X2
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|-
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!scope="row"| H2O
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| 40
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| 80
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|-
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!scope="row"| dNTPs
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| 1
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| 2
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|-
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!scope="row"| F primer
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| 1
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| 2
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|-
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!scope="row"| R primer
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| 1
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| 2
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|-
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!scope="row"| DNA
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| 1
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| 2*1
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|-
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!scope="row"| Pfu X10 buffer
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| 5
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| 10
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|-
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!scope="row"| Pfu turbo pol
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| 1
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| 2
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|}
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:::::::{| border="0" width="50%" |
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! align="left" | MITF
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! align="left" | yCCS
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! conditions
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|-
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|MITF_Site1_F
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|yCCS_F
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!align="left" width="1%"|time
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!align="left" width="1%"|°C
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|-
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|MITF_Site2_R
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|yCCS_R
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|30s
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|95
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|-
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|pRc/CMV
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|pSB1C3
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|30s
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|95
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|-
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|~6.7kb
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|~3,5kb
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|30s
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|55
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|-
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|
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|
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|7m
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|68
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|-
 +
|
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|
 +
|oo
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|4
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|}
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 +
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::*Make sure sample is ≤ 37°C
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::*Add 1µl Dpn1 (to 50µl product) and incubate in 37°C ON
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 +
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===Transformation===
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::<u>mix</u>
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::Top 10 competent cells 100µl
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::pRc/CMV.MITF_M 1µl
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*Hold on ice 30min
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*Heat shock 42&degC, 55sec
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*Cool down 1min (on ice)
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*Add 900µl LB
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*Incubate 37&deg;C, 250rpm, 1h (forgot rpm)
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*Spinn down cells, 13000rpm, 15sec
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*Remove 900µl LB
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*Plate the 100µl on a Amp-plate
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*Grow ON at 37&deg;C
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 +
 +
 +
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==Hassan==
==Hassan==

Revision as of 18:30, 2 August 2010

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Contents

Mimmi

over-expression in pEX

- remaking the comassie-gel


Mix v
LB 4 ml
glycose 40 µl (1%)
Amp 8 µl (0.5%)
old culture 40 µl


  • Grow in 37C, ~200 rpm ~2h until OD = 0.6
  • Add IPTG (1 µl/ml,1M)in one of the two cultures, the other one is used as a control
  • Take sample at 0h, 1h, 2h, 3h
    • pipette 500µl into a 1.5ml tube
    • spinn down the sells and remove LB
    • resuspend in 50µl loading dye
    • freeze


Site-Directed Mutagenesis

Primers

(MW/10)/(OD*33) = dilution factor
1000/df = volyme to get 100µM primer
100µM = (MW/10) ng/µl
(MW/10)/125 = df to get 125 ng/µl
primer V H2O (µl) V H2O to get 125 ng/µl (µl)
MITF_site1_F 463.88 3µl + 15.4µl
MITF_site1_R 64.77 3µl + 15.3µl
MITF_site2_F 144.85 3µl + 21µl
MITF_site2_R 141.77 3µl + 21.6µl
. concentration .
yCCS_F 1085.7 ng/µl 3µl + 23µl
yCCS_R 996.5 ng/µl 3µl + 20.9µl

reaction

Mix (µl) X2
H2O 40 80
dNTPs 1 2
F primer 1 2
R primer 1 2
DNA 1 2*1
Pfu X10 buffer 5 10
Pfu turbo pol 1 2
MITF yCCS conditions
MITF_Site1_F yCCS_F time °C
MITF_Site2_R yCCS_R 30s 95
pRc/CMV pSB1C3 30s 95
~6.7kb ~3,5kb 30s 55
7m 68
oo 4


  • Make sure sample is ≤ 37°C
  • Add 1µl Dpn1 (to 50µl product) and incubate in 37°C ON


Transformation

mix
Top 10 competent cells 100µl
pRc/CMV.MITF_M 1µl


  • Hold on ice 30min
  • Heat shock 42&degC, 55sec
  • Cool down 1min (on ice)
  • Add 900µl LB
  • Incubate 37°C, 250rpm, 1h (forgot rpm)
  • Spinn down cells, 13000rpm, 15sec
  • Remove 900µl LB
  • Plate the 100µl on a Amp-plate
  • Grow ON at 37°C




Hassan

Jensen et al. Nucleic Acids Res. 2009, 37(Database issue):D412-6
Jensen et al. Nucleic Acids Res. 2009, 37(Database issue):D412-6

[http://http://string.embl.de/version_8_3/newstring_cgi/show_network_section.pl?identifiers=9606.ENSP00000331746%250D9606.ENSP00000295600%250D9606.ENSP00000364016%250D9606.ENSP00000371175%250D9606.ENSP00000269280%250D9606.ENSP00000363700%250D9606.ENSP00000360157%250D9606.ENSP00000373571%250D9606.ENSP00000291700&channel1=off&channel2=off&channel3=off&channel4=on&channel5=on&channel6=on&channel7=on&interactive=yes&network_flavor=actions&targetmode=proteins]

[http://http://string.embl.de/version_8_3/newstring_cgi/show_network_section.pl?identifiers=9606.ENSP00000331746%250D9606.ENSP00000295600%250D9606.ENSP00000226877%250D9606.ENSP00000371175%250D9606.ENSP00000269280%250D9606.ENSP00000363700%250D9606.ENSP00000360157%250D9606.ENSP00000373571%250D9606.ENSP00000291700&channel1=off&channel2=off&channel3=off&channel4=on&channel5=on&channel6=on&channel7=on&additional_network_nodes=5&interactive=yes&network_flavor=actions&targetmode=proteins]









































Nina

Colony PCR on IgG protease in shipping vector

I made a colony PCR on the IgG protease that I have inserted into the iGEM shipping vector to verify that the gene is inserted in a correct position. Therefore I used one of the gene's primers and one of the vector's verification primers. The colony numbers are: 1, 2, 3, 4 and 5.

PCR reaction mix:

  • 1 µl Morten's polymerase PjuX7
  • 1 µl 10 mM dNTPs
  • 3 µl 5 µM forward primer (VF2)
  • 3 µl 5 µM revers primer (gene's primer)
  • 10 µl buffer 5X
  • 1 µl MgCl2 50mM
  • 30 µl H2O
  • DNA template was one colony

PCR program:

98°C - 2 min

31 cycles of:

  • 98°C - 10 sec
  • 55°C - 15 sec
  • 72°C - 1.5 min

72°C - 5 min

4°C - ∞

Igg-shipping.jpg

DNA Ladder: FastRuler™ Middle Range, ready-to-use, 100-5000 bp Fermentas

Colony number 2 looks good on the gel. This one will be inoculated in LB in order to become minipreped to be shipped to iGEM hq.

Mini prep on IgG protease and CPP

I prepare a miniprep on the inoculated IgG protease with colony number 2. In addition I miniprep three inoculated samples with vector carrying CPP from colony number 22, 23 and 33.

The method is carried out according to the procedure in protocols.

Measuring concentration with spectrophotometer:

Spek.jpg

Sequencing CPP TAT N version

I send three samples of CPP TAT N version for sequencing. Colony numbers are: 22, 23 and 33.

  • 15 ul vector
  • 1.5 ul 10uM VR2 primer

22: ASB0045 105

23: ASB0045 104

33: ASB0045 103

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