Team:Stockholm/20 August 2010
From 2010.igem.org
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==Andreas== | ==Andreas== | ||
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- | + | ===Transformation results=== | |
+ | ''From 19/8 transformations'' | ||
*pEX.RFP: Good yield of red (positive) and white (negative) colonies. | *pEX.RFP: Good yield of red (positive) and white (negative) colonies. | ||
*pSB1K3.SOD⋅His: Very few colonies | *pSB1K3.SOD⋅His: Very few colonies | ||
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====Colony PCR of pEX.RFP, pEX and pMA.His==== | ====Colony PCR of pEX.RFP, pEX and pMA.His==== | ||
Picked four pEX.RFP colonies for PCR verification. Also ran PCR on the pEX and pMA.His clones from 19/8. PCR and gel run by Mimmi (see above). | Picked four pEX.RFP colonies for PCR verification. Also ran PCR on the pEX and pMA.His clones from 19/8. PCR and gel run by Mimmi (see above). | ||
+ | |||
=====Results===== | =====Results===== | ||
*pMA.His clone verified for correct insert. | *pMA.His clone verified for correct insert. | ||
*pEX and pEX.RFP need to be re-run, as they did not result in any bands. | *pEX and pEX.RFP need to be re-run, as they did not result in any bands. | ||
- | ====Plasmid prep | + | ====ON cultures==== |
+ | *pMA.His | ||
+ | *pEX (not yet verified) | ||
+ | *pEX.RFP 3 (not yet verified) | ||
+ | |||
+ | 3 ml LB + 100 Amp. 30 °C ON. | ||
+ | |||
+ | ===Plasmid prep=== | ||
''From 19/8 ON cultures'' | ''From 19/8 ON cultures'' | ||
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|} | |} | ||
- | + | ===Assembly of His⋅SOD/yCCS into pSB1K3=== | |
''Step I of C-CPP cloning strategy (19/8)'' | ''Step I of C-CPP cloning strategy (19/8)'' | ||
- | + | ====Digestions==== | |
[pSB1C3.m-yCCS 1] = 101.2 ng/μl<br /> | [pSB1C3.m-yCCS 1] = 101.2 ng/μl<br /> | ||
[pSB1C3.m-SOD 2] = 105.5 ng/μl | [pSB1C3.m-SOD 2] = 105.5 ng/μl | ||
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† ''Miscalculation'' | † ''Miscalculation'' | ||
- | + | ====Ligations==== | |
Without prior enzyme inactivation or DNA purification. | Without prior enzyme inactivation or DNA purification. | ||
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Enzyme inactivation (not PstI): 80 °C, 20 min. | Enzyme inactivation (not PstI): 80 °C, 20 min. | ||
- | + | ====Transformation==== | |
Standard transformation protocol. | Standard transformation protocol. | ||
*1 μl ligation mix | *1 μl ligation mix | ||
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**Lig pSB1K3.His⋅yCCS | **Lig pSB1K3.His⋅yCCS | ||
*Cells plated onto 50 Km LB agar plates | *Cells plated onto 50 Km LB agar plates | ||
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Revision as of 22:35, 23 August 2010
Contents |
Hassan
midnight update, after a fast recovery from sickness!
Version 0.5.1
Version 0.6.1
Mimmi
pMA
Primer dilution
pMA_VF | 91µl sH2O --> 100µM | 10µl + 90µl sH2O --> 10µM | ||
pMA_VR | 91µl sH2O --> 100µM | 10µl + 90µl sH2O --> 10µM |
pMA/pEX
verification PCR
pEX | pMA | ||||||||
---|---|---|---|---|---|---|---|---|---|
Mix | (µl) | X6 | X3 | primers | conditions | ||||
sH2O | 22.5 | 135 | 67.5 | pEX_VF | time | °C | |||
F primer | 1 | 6 | 3 | pEX_VR | 2m | 94 | |||
R primer | 1 | 6 | 3 | pMA_VF | 30s | 94 | ) | ||
DNA | 0.5 | 5X0.5 | 2X0.5 | pMA_VR | 30s | 60 | > 30 cycles | ||
tot | 25µl | 250µl | 75µl | 2m30s | 72 | ) | |||
10m | 72 | ||||||||
oo | 10 |
Andreas
Transformation results
From 19/8 transformations
- pEX.RFP: Good yield of red (positive) and white (negative) colonies.
- pSB1K3.SOD⋅His: Very few colonies
- pSB1K3.yCCS.His: Very few colonies
- Km probably not feasible with quick transformation.
Colony PCR of pEX.RFP, pEX and pMA.His
Picked four pEX.RFP colonies for PCR verification. Also ran PCR on the pEX and pMA.His clones from 19/8. PCR and gel run by Mimmi (see above).
Results
- pMA.His clone verified for correct insert.
- pEX and pEX.RFP need to be re-run, as they did not result in any bands.
ON cultures
- pMA.His
- pEX (not yet verified)
- pEX.RFP 3 (not yet verified)
3 ml LB + 100 Amp. 30 °C ON.
Plasmid prep
From 19/8 ON cultures
- E.Z.N.A. Plasmid Mini Prep kit.
- 70 μl elution volume
DNA concentrations | ||
---|---|---|
Sample | Conc. [ng/μl] | A260/A280 |
pSB1K3.BBa_J04450 | 103.8 | 1.94 |
pEX | 55.52 | 1.89 |
pMA.His | 85.67 | 1.87 |
Assembly of His⋅SOD/yCCS into pSB1K3
Step I of C-CPP cloning strategy (19/8)
Digestions
[pSB1C3.m-yCCS 1] = 101.2 ng/μl
[pSB1C3.m-SOD 2] = 105.5 ng/μl
[μl] | pMA.His | pSB1C3.m-yCCS 1 | pSB1C3.m-SOD 1 | Inc.: 37 °C, 0:30 (FD) or 1:30 (NgoMIV) |
---|---|---|---|---|
10X FD buffer | 3 | 3 | 3 | |
dH2O | 6.7† | 6 | 7 | |
2 μg DNA | 23.3 | 20 | 19 | |
FD EcoRI | 0.5 | – | – | |
FD PstI | – | 1 | 1 | |
FD AgeI | 0.5 | – | – | |
NgoMIV | – | 1 | 1 | |
30† | 30 | 30 |
† Miscalculation
Ligations
Without prior enzyme inactivation or DNA purification.
[μl] | Lig pSB1K3. His⋅SOD | Lig pSB1K3. His⋅yCCS | Vector/insert ratio: 1:3. Inc.: 22 °C, 0:10 |
---|---|---|---|
100 ng vector | 2.2 | 2.2 | |
His insert | 4.0 | 4.0 | |
SOD insert | 5.1 | – | |
yCCS | – | 5.7 | |
5X Rapid Lig. buf. | 4 | 4 | |
dH2O | 2.7 | 2.1 | |
T4 DNA ligase | 1 | 1 | |
20 | 20 |
Enzyme inactivation (not PstI): 80 °C, 20 min.
Transformation
Standard transformation protocol.
- 1 μl ligation mix
- Lig pSB1K3.His⋅SOD
- Lig pSB1K3.His⋅yCCS
- Cells plated onto 50 Km LB agar plates