Team:Stockholm/10 August 2010

From 2010.igem.org

(Difference between revisions)
(→Mimmi)
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==Andreas==
==Andreas==
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 +
===Cloning of IgG protease===
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''Continued from 9/8 ON plates''
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====Colony PCR====
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Four clones (A, B, C, D) were picked for colony PCR. Positive control: pSB1C3.BBa_J04450 plasmid.
 +
Procedures according to protocol; 1:40 elongation time.
 +
 +
====Gel verification====
 +
 +
1 % agarose, 90 V, 40 min
 +
 +
'''Results:''' Weak and very irregularly sized bands. Probably something wrong with the ligation mixture. New IgGp digestion will be made.
 +
 +
===New IgG protease cloning===
 +
 +
====Digestion====
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Used pSB1A3.IgGp at a conc. of 139 ng/μl.
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 +
{|border="1" cellpadding="2" cellspacing="0"
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|'''10X FD buffer'''
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|2 μl
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|-
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|'''dH<sub>2</sub>O'''
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|3 &mu;l
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|-
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|'''2 &mu;g DNA'''
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|14 &mu;l
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|-
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|'''FD SpeI'''
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|0.5 &mu;l
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|-
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|'''FD EcoRI'''
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|0.5 &mu;l
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|-
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|&nbsp;
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|20 &mu;l
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|}
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Incubation: 37 &deg;C, 30 min

Revision as of 13:56, 11 August 2010

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Contents

Mimmi

yCCS and SOD

redoing the Site-Directed Mutagenesis

Mix (µl) X2 X2 yCCS A SOD A conditions
sH2O 40 80 yCCS_mut_F SOD_mut_F time °C
dNTPs 1 2 yCCS_mut_R SOD_mut_R 30s 95
F primer 1 2 pSB1C3 pSB1C3 30s 95 )
R primer 1 2 ~3.5kb ~3.2kb 30s 55 > 22 cycles
DNA 1 2X1 4m 68 )
Pfu buffer 5 10 oo 10
Pfu turbo 1 2
tot 50
SOD Primers
SOD_mut_F 262.55µl H2O --> 100µM
SOD_mut_R 372.64µl H2O --> 100µM
SOD_mut_F 3µl + 24.15µl H2O --> 125ng/µl
SOD_mut_R 3µl + 24.42µl H2O --> 125ng/µl


MITF

Amplifying

Mix phusion (µl) /4 Mix Pfu turbo (µl) /4 Primers
sH2O 67 sH2O 77 MITF_F
F primer 5 F primer 5 MITF_R
R primer 5 R primer 5
dNTP 2 dNTP 2 conditions
5X buffer 20 10X buffer 10 time °C
Phusion pol. 1 Pfu pol. 1 2m 98
tot 100 25 tot 100 25 30s 98 )
30s 40-55 > 5 cycles
1m30s 72 )
30s 98 )
30s 65 > 25 cycles
1m30s 72 )
10m 72
oo 10

Andreas

Cloning of IgG protease

Continued from 9/8 ON plates

Colony PCR

Four clones (A, B, C, D) were picked for colony PCR. Positive control: pSB1C3.BBa_J04450 plasmid. Procedures according to protocol; 1:40 elongation time.

Gel verification

1 % agarose, 90 V, 40 min

Results: Weak and very irregularly sized bands. Probably something wrong with the ligation mixture. New IgGp digestion will be made.

New IgG protease cloning

Digestion

Used pSB1A3.IgGp at a conc. of 139 ng/μl.

10X FD buffer 2 μl
dH2O 3 μl
2 μg DNA 14 μl
FD SpeI 0.5 μl
FD EcoRI 0.5 μl
  20 μl

Incubation: 37 °C, 30 min

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