Team:Stockholm/10 August 2010
From 2010.igem.org
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==Andreas== | ==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. | ||
+ | |||
+ | {|border="1" cellpadding="2" cellspacing="0" | ||
+ | |'''10X FD buffer''' | ||
+ | |2 μl | ||
+ | |- | ||
+ | |'''dH<sub>2</sub>O''' | ||
+ | |3 μl | ||
+ | |- | ||
+ | |'''2 μg DNA''' | ||
+ | |14 μl | ||
+ | |- | ||
+ | |'''FD SpeI''' | ||
+ | |0.5 μl | ||
+ | |- | ||
+ | |'''FD EcoRI''' | ||
+ | |0.5 μl | ||
+ | |- | ||
+ | | | ||
+ | |20 μl | ||
+ | |} | ||
+ | |||
+ | Incubation: 37 °C, 30 min |
Revision as of 13:56, 11 August 2010
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