Team:Stockholm/1 September 2010
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==Andreas== | ==Andreas== | ||
+ | |||
+ | ===His⋅SOD cloning into pMA=== | ||
+ | |||
+ | ====Digestion==== | ||
+ | |||
+ | [pSB1C3.m-SOD]=105.5 ng/μl<br /> | ||
+ | [pMA.His]=85.67 ng/μl | ||
+ | |||
+ | {|border="1" cellpadding="1" cellspacing="0" | ||
+ | |align="center"|[μl] | ||
+ | !width="50"|SOD | ||
+ | !width="50"|pMA.His | ||
+ | |- | ||
+ | |10X FD buffer | ||
+ | |align="center"|3 | ||
+ | |align="center"|3 | ||
+ | |- | ||
+ | |DNA (2 μg) | ||
+ | |align="center"|19 | ||
+ | |align="center"|23 | ||
+ | |- | ||
+ | |dH<sub>2</sub>O | ||
+ | |align="center"|6 | ||
+ | |align="center"|3 | ||
+ | |- | ||
+ | |FD AgeI | ||
+ | |align="center"|0 | ||
+ | |align="center"|0.5 | ||
+ | |- | ||
+ | |NgoMIV | ||
+ | |align="center"|1 | ||
+ | |align="center"|0 | ||
+ | |- | ||
+ | |FD SpeI | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |- | ||
+ | | | ||
+ | !30 | ||
+ | !30.5 | ||
+ | |} | ||
+ | |||
+ | '''Incubation:''' | ||
+ | *37 °C, 0:30 (FD) | ||
+ | *37 °C, 2:00 (NgoMIV) | ||
+ | |||
+ | '''Inactivation:''' | ||
+ | *80 °C, 10 min | ||
+ | |||
+ | ====Ligation==== | ||
+ | |||
+ | {|border="1" cellpadding="1" cellspacing="1" | ||
+ | | | ||
+ | |align="center"|[μl] | ||
+ | |- | ||
+ | |5X Rapid ligation buf. | ||
+ | |width="40" align="center"|4 | ||
+ | |- | ||
+ | |Vector DNA (pMa.His) | ||
+ | |align="center"|1.5 | ||
+ | |- | ||
+ | |Insert DNA (m-SOD) | ||
+ | |align="center"|4.5 | ||
+ | |- | ||
+ | |dH<sub>2</sub>O | ||
+ | |align="center"|9 | ||
+ | |- | ||
+ | |T4 DNA ligase | ||
+ | |align="center"|1 | ||
+ | |- | ||
+ | | | ||
+ | !20 | ||
+ | |} | ||
+ | |||
+ | '''Incubation:''' | ||
+ | *22 °C, 10 min | ||
+ | |||
+ | ====Transformation==== | ||
+ | Quick-transformation according to protocol into both Top10 and DH5α to compare and verify Top10 competence: | ||
+ | *1.5 μl | ||
+ | *Amp 100 | ||
+ | |||
+ | ===SOD⋅His cloning into pMA=== | ||
+ | |||
+ | ====Glycerol stocks==== | ||
+ | ''From 31/8 ON cultures'' | ||
+ | |||
+ | *SH1: pMA.SOD⋅His 1, 1/9/2010 | ||
+ | *SH2: pMA.SOD⋅His 2, 1/9/2010 | ||
+ | |||
+ | ===Transfer of m-yCCS into pEX=== | ||
+ | |||
+ | ====Digestion==== | ||
+ | [pEX.RFP] = 82 ng/μl | ||
+ | [pSB1C3.m-yCCS] = 101.2 ng/μl | ||
+ | |||
+ | {|border="1" cellpadding="1" cellspacing="0" | ||
+ | |align="center"|[μl] | ||
+ | !width="50"|SOD | ||
+ | !width="50"|pMA.His | ||
+ | |- | ||
+ | |10X FD buffer | ||
+ | |align="center"|3 | ||
+ | |align="center"|3 | ||
+ | |- | ||
+ | |DNA (1 μg) | ||
+ | |align="center"|12 | ||
+ | |align="center"|6 | ||
+ | |- | ||
+ | |dH<sub>2</sub>O | ||
+ | |align="center"|13 | ||
+ | |align="center"|19 | ||
+ | |- | ||
+ | |FD XbaI | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |- | ||
+ | |FD PstI | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |- | ||
+ | | | ||
+ | !30 | ||
+ | !30 | ||
+ | |} | ||
+ | |||
+ | '''Incubation:''' | ||
+ | *37 °C, 10 min | ||
+ | |||
+ | '''Inactivation:''' | ||
+ | *65 °C, 20 min | ||
+ | |||
+ | ====Ligation==== | ||
+ | |||
+ | {|border="1" cellpadding="1" cellspacing="1" | ||
+ | | | ||
+ | |align="center"|[μl] | ||
+ | |- | ||
+ | |5X Rapid ligation buf. | ||
+ | |width="40" align="center"|4 | ||
+ | |- | ||
+ | |Vector DNA (pEX) | ||
+ | |align="center"|1.5 | ||
+ | |- | ||
+ | |Insert DNA (m-yCCS) | ||
+ | |align="center"|9 | ||
+ | |- | ||
+ | |dH<sub>2</sub>O | ||
+ | |align="center"|4.5 | ||
+ | |- | ||
+ | |T4 DNA ligase | ||
+ | |align="center"|1 | ||
+ | |- | ||
+ | | | ||
+ | !20 | ||
+ | |} | ||
+ | |||
+ | '''Incubation:''' | ||
+ | *22 °C, 10 min | ||
+ | |||
+ | ====Transformation==== | ||
+ | Quick-transformation (Top10) | ||
+ | *2 μl | ||
+ | *Amp 100 |
Revision as of 21:57, 3 September 2010
Contents |
Andreas
His⋅SOD cloning into pMA
Digestion
[pSB1C3.m-SOD]=105.5 ng/μl
[pMA.His]=85.67 ng/μl
[μl] | SOD | pMA.His |
---|---|---|
10X FD buffer | 3 | 3 |
DNA (2 μg) | 19 | 23 |
dH2O | 6 | 3 |
FD AgeI | 0 | 0.5 |
NgoMIV | 1 | 0 |
FD SpeI | 1 | 1 |
30 | 30.5 |
Incubation:
- 37 °C, 0:30 (FD)
- 37 °C, 2:00 (NgoMIV)
Inactivation:
- 80 °C, 10 min
Ligation
[μl] | |
5X Rapid ligation buf. | 4 |
Vector DNA (pMa.His) | 1.5 |
Insert DNA (m-SOD) | 4.5 |
dH2O | 9 |
T4 DNA ligase | 1 |
20 |
---|
Incubation:
- 22 °C, 10 min
Transformation
Quick-transformation according to protocol into both Top10 and DH5α to compare and verify Top10 competence:
- 1.5 μl
- Amp 100
SOD⋅His cloning into pMA
Glycerol stocks
From 31/8 ON cultures
- SH1: pMA.SOD⋅His 1, 1/9/2010
- SH2: pMA.SOD⋅His 2, 1/9/2010
Transfer of m-yCCS into pEX
Digestion
[pEX.RFP] = 82 ng/μl [pSB1C3.m-yCCS] = 101.2 ng/μl
[μl] | SOD | pMA.His |
---|---|---|
10X FD buffer | 3 | 3 |
DNA (1 μg) | 12 | 6 |
dH2O | 13 | 19 |
FD XbaI | 1 | 1 |
FD PstI | 1 | 1 |
30 | 30 |
Incubation:
- 37 °C, 10 min
Inactivation:
- 65 °C, 20 min
Ligation
[μl] | |
5X Rapid ligation buf. | 4 |
Vector DNA (pEX) | 1.5 |
Insert DNA (m-yCCS) | 9 |
dH2O | 4.5 |
T4 DNA ligase | 1 |
20 |
---|
Incubation:
- 22 °C, 10 min
Transformation
Quick-transformation (Top10)
- 2 μl
- Amp 100