Team:Stockholm/8 September 2010

From 2010.igem.org

(Difference between revisions)
(New page: {{Stockholm/Top2}} ==Andreas==)
(→Andreas)
Line 2: Line 2:
==Andreas==
==Andreas==
 +
===Transfer of yCCS into pEX===
 +
====Glycerol stocks====
 +
''From 7/9 ON cultures''
 +
*pEX.yCCS 5 2010-09-08
 +
*pEX.yCCS 8 2010-09-08
 +
 +
====Plasmid prep====
 +
''From 7/9 ON cultures''<br />
 +
Elution: 70 &mu;l x2
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
!Sample
 +
!Conc [ng/&mu;l]
 +
!A<sub>260</sub>/A<sub>280</sub>
 +
|-
 +
|pEX.yCCS 5
 +
|align="center"|56.32
 +
|align="center"|2.02
 +
|-
 +
|pEX.yCCS 8
 +
|align="center"|57.63
 +
|align="center"|1.97
 +
|}
 +
 +
===Cloning of N-CPPs into pSB1C3===
 +
====Plasmid prep====
 +
''From 7/9 ON cultures''<br />
 +
Elution: 70 &mu;l x2
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
!Sample
 +
!Conc [ng/&mu;l]
 +
!A<sub>260</sub>/A<sub>280</sub>
 +
|-
 +
|pSB1C3.N-CPP 3
 +
|align="center"|105.6
 +
|align="center"|1.94
 +
|-
 +
|pSB1C3.N-CPP 4
 +
|align="center"|88.87
 +
|align="center"|2.03
 +
|-
 +
|pSB1C3.N-CPP 6*
 +
|align="center"|86.64
 +
|align="center"|1.98
 +
|-
 +
|pSB1C3.N-CPP 7*
 +
|align="center"|75.79
 +
|align="center"|1.90
 +
|-
 +
|pSB1C3.N-CPP 8*
 +
|align="center"|52.64
 +
|align="center"|1.99
 +
|}
 +
 +
====Sequencing====
 +
''15 &mu;l plasmid DNA + 1.5 &mu;l VF2 primer.''
 +
*'''NCPP 3:''' ASB0045 B41
 +
*'''NCPP 4:''' ASB0045 B42
 +
*'''NCPP 6:''' ASB0045 B43
 +
*'''NCPP 7:''' ASB0045 B44
 +
*'''NCPP 8:''' ASB0045 B45
 +
 +
===Construction and cloning of MITF===
 +
====MITF PCR====
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
!colspan="2"|PCR tubes (MITF 1 & MITF 2)
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center" width="50"|41
 +
|-
 +
|Pfu buffer
 +
|align="center"|5
 +
|-
 +
|dNTPs, 10 mM
 +
|align="center"|1
 +
|-
 +
|Fwd primer<br />(MITF_FB_F_18aug)
 +
|align="center"|1
 +
|-
 +
|Rev primer<br />(MITF_FB_R_18aug)
 +
|align="center"|1
 +
|-
 +
|Template DNA<br />(pRC/CMV MITF-M, 10 ng/&mu;l)
 +
|align="center"|0.5
 +
|-
 +
|Pfu polymerase
 +
|align="center"|0.5
 +
|-
 +
|&nbsp;
 +
!50 &mu;l
 +
|}
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
!colspan="3"|PCR settings
 +
|-
 +
|width="50"|95 &deg;C
 +
|align="center" colspan="2"|10:00
 +
|-
 +
|95 &deg;C
 +
|width="50" align="center"|0:30
 +
|rowspan="3"|x5
 +
|-
 +
|55 &deg;C
 +
|align="center"|0:30
 +
|-
 +
|72 &deg;C
 +
|align="center"|1:40
 +
|-
 +
|95 &deg;C
 +
|align="center"|0:30
 +
|rowspan="2"|x25
 +
|-
 +
|72 &deg;C
 +
|align="center"|2:10
 +
|-
 +
|72 &deg;C
 +
|align="center" colspan="2"|10:00
 +
|}
 +
 +
====Gel verification====
 +
1 % agarose, 110 V
 +
 +
*3 &mu;l DNA ladder (O'GeneRuler 1 kb DNA ladder
 +
*5 &mu;l sample
 +
 +
''No gel image available''
 +
 +
'''Results'''<br />
 +
No visible bands for neither of the two MITF samples.
 +
 +
===Colony PCRs===
 +
Picked colonies from Mimmi's plates:
 +
*pSB1C3.MITF (2/9)
 +
**MITF 1-8
 +
*pSB1C3.SOD&sdot;His (6/9)
 +
**pC.SH 1-3
 +
*pSB1C3.His&sdot;SOD (6/9)
 +
**pC.HS 1-3
 +
*pEX.His&sdot;SOD (6/9)
 +
**pEX.SH 1-3
 +
*pEX.SOD&sdot;His (6/9)
 +
**pEX.HS 1-3
 +
 +
'''Primers'''
 +
*pSB1C3: VF2 & VR
 +
*pEX: pEXf & pEXr
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
!colspan="2"|PCR tubes
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center" width="50"|16.22
 +
|-
 +
|DreamTaq buffer
 +
|align="center"|2
 +
|-
 +
|dNTPs, 10 mM
 +
|align="center"|0.4
 +
|-
 +
|DreamTaq pol.
 +
|align="center"|0.08
 +
|-
 +
|Fwd primer
 +
|align="center"|0.4
 +
|-
 +
|Rev primer
 +
|align="center"|0.4
 +
|-
 +
|Cell susp. (template)
 +
|align="center"|0.5
 +
|-
 +
|&nbsp;
 +
!20 &mu;l
 +
|}
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
!colspan="3"|PCR settings
 +
|-
 +
|width="50"|95 &deg;C
 +
|align="center" colspan="2"|3:00
 +
|-
 +
|95 &deg;C
 +
|width="50" align="center"|0:30
 +
|rowspan="3"|x30
 +
|-
 +
|55 &deg;C
 +
|align="center"|0:30
 +
|-
 +
|72 &deg;C
 +
|align="center"|1:00
 +
|-
 +
|72 &deg;C
 +
|align="center" colspan="2"|5:00
 +
|}
 +
 +
====Gel verification====
 +
1 % agarose, 90 V, 50 min
 +
 +
'''Expected bands'''
 +
*MITF: 1586 bp
 +
*pC.SH/pC.HS: 815 bp
 +
*pEX.SH/pEX.HS: 702 bp
 +
 +
'''Results'''<br />

Revision as of 17:14, 8 September 2010

Home Project Idea Lab Work Results Modelling Team       Follow us on and        2010.igem.org

↳   Notebook Safety Protocols


Contents

Andreas

Transfer of yCCS into pEX

Glycerol stocks

From 7/9 ON cultures

  • pEX.yCCS 5 2010-09-08
  • pEX.yCCS 8 2010-09-08

Plasmid prep

From 7/9 ON cultures
Elution: 70 μl x2

Sample Conc [ng/μl] A260/A280
pEX.yCCS 5 56.32 2.02
pEX.yCCS 8 57.63 1.97

Cloning of N-CPPs into pSB1C3

Plasmid prep

From 7/9 ON cultures
Elution: 70 μl x2

Sample Conc [ng/μl] A260/A280
pSB1C3.N-CPP 3 105.6 1.94
pSB1C3.N-CPP 4 88.87 2.03
pSB1C3.N-CPP 6* 86.64 1.98
pSB1C3.N-CPP 7* 75.79 1.90
pSB1C3.N-CPP 8* 52.64 1.99

Sequencing

15 μl plasmid DNA + 1.5 μl VF2 primer.

  • NCPP 3: ASB0045 B41
  • NCPP 4: ASB0045 B42
  • NCPP 6: ASB0045 B43
  • NCPP 7: ASB0045 B44
  • NCPP 8: ASB0045 B45

Construction and cloning of MITF

MITF PCR

PCR tubes (MITF 1 & MITF 2)
dH2O 41
Pfu buffer 5
dNTPs, 10 mM 1
Fwd primer
(MITF_FB_F_18aug) 		1
Rev primer
(MITF_FB_R_18aug) 		1
Template DNA
(pRC/CMV MITF-M, 10 ng/μl) 		0.5
Pfu polymerase 0.5
  50 μl
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

Gel verification

1 % agarose, 110 V

  • 3 μl DNA ladder (O'GeneRuler 1 kb DNA ladder
  • 5 μl sample

No gel image available

Results
No visible bands for neither of the two MITF samples.

Colony PCRs

Picked colonies from Mimmi's plates:

  • pSB1C3.MITF (2/9)
    • MITF 1-8
  • pSB1C3.SOD⋅His (6/9)
    • pC.SH 1-3
  • pSB1C3.His⋅SOD (6/9)
    • pC.HS 1-3
  • pEX.His⋅SOD (6/9)
    • pEX.SH 1-3
  • pEX.SOD⋅His (6/9)
    • pEX.HS 1-3

Primers

  • pSB1C3: VF2 & VR
  • pEX: pEXf & pEXr
PCR tubes
dH2O 16.22
DreamTaq buffer 2
dNTPs, 10 mM 0.4
DreamTaq pol. 0.08
Fwd primer 0.4
Rev primer 0.4
Cell susp. (template) 0.5
  20 μl
PCR settings
95 °C 3:00
95 °C 0:30 x30
55 °C 0:30
72 °C 1:00
72 °C 5:00

Gel verification

1 % agarose, 90 V, 50 min

Expected bands

  • MITF: 1586 bp
  • pC.SH/pC.HS: 815 bp
  • pEX.SH/pEX.HS: 702 bp

Results

Retrieved from "http://2010.igem.org/Team:Stockholm/8_September_2010"