Team:Stockholm/20 August 2010
From 2010.igem.org
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==Andreas== | ==Andreas== | ||
+ | ===Assembly of CPP⋅protein⋅His constructs=== | ||
+ | ''Continued from 19/8'' | ||
+ | |||
+ | ====Transformation results==== | ||
+ | *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. | ||
+ | |||
+ | ====Plasmid prep==== | ||
+ | ''From 19/8 ON cultures'' | ||
+ | |||
+ | *E.Z.N.A. Plasmid Mini Prep kit. | ||
+ | *70 μl elution volume | ||
+ | |||
+ | {| border="1" cellpadding="1" cellspacing="0" | ||
+ | !colspan="3"|DNA concentrations | ||
+ | |- | ||
+ | !Sample | ||
+ | !Conc. [ng/μl] | ||
+ | !A<sub>260</sub>/A<sub>280</sub> | ||
+ | |- | ||
+ | |pSB1K3.BBa_J04450 | ||
+ | |align="center"|103.8 | ||
+ | |align="center"|1.94 | ||
+ | |- | ||
+ | |pEX | ||
+ | |align="center"|55.52 | ||
+ | |align="center"|1.89 | ||
+ | |- | ||
+ | |pMA.His | ||
+ | |align="center"|85.67 | ||
+ | |align="center"|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 | ||
+ | |||
+ | {|border="1" cellpadding="1" cellspacing="0" | ||
+ | |align="center"|[μl] | ||
+ | !width="120"|pMA.His | ||
+ | !width="120"|pSB1C3.m-yCCS 1 | ||
+ | !width="120"|pSB1C3.m-SOD 1 | ||
+ | |rowspan="9" width="120" align="center"|Inc.: 37 °C,<br /> 0:30 (FD)<br /> or<br /> 1:30 (NgoMIV) | ||
+ | |- | ||
+ | |'''10X FD buffer''' | ||
+ | |align="center"|3 | ||
+ | |align="center"|3 | ||
+ | |align="center"|3 | ||
+ | |- | ||
+ | |'''dH<sub>2</sub>O''' | ||
+ | |align="center"|6.7† | ||
+ | |align="center"|6 | ||
+ | |align="center"|7 | ||
+ | |- | ||
+ | |'''2 μg DNA''' | ||
+ | |align="center"|23.3 | ||
+ | |align="center"|20 | ||
+ | |align="center"|19 | ||
+ | |- | ||
+ | |'''FD EcoRI''' | ||
+ | |align="center"|0.5 | ||
+ | |align="center"|– | ||
+ | |align="center"|– | ||
+ | |- | ||
+ | |'''FD PstI''' | ||
+ | |align="center"|– | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |- | ||
+ | |'''FD AgeI''' | ||
+ | |align="center"|0.5 | ||
+ | |align="center"|– | ||
+ | |align="center"|– | ||
+ | |- | ||
+ | |'''NgoMIV''' | ||
+ | |align="center"|– | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |- | ||
+ | | | ||
+ | |align="center"|'''30'''† | ||
+ | |align="center"|'''30''' | ||
+ | |align="center"|'''30''' | ||
+ | |} | ||
+ | † ''Miscalculation'' | ||
+ | |||
+ | =====Ligations===== | ||
+ | Without prior enzyme inactivation or DNA purification. | ||
+ | |||
+ | {|border="1" cellpadding="1" cellspacing="0" | ||
+ | |align="center"|[μl] | ||
+ | !Lig pSB1K3.<br />His⋅SOD | ||
+ | !Lig pSB1K3.<br />His⋅yCCS | ||
+ | |rowspan="9" align="center"|Vector/insert ratio:<br />1:3.<br /><br /> Inc.: 22 °C, 0:10 | ||
+ | |- | ||
+ | |'''100 ng vector''' | ||
+ | |align="center"|2.2 | ||
+ | |align="center"|2.2 | ||
+ | |- | ||
+ | |'''His insert''' | ||
+ | |align="center"|4.0 | ||
+ | |align="center"|4.0 | ||
+ | |- | ||
+ | |'''SOD insert''' | ||
+ | |align="center"|5.1 | ||
+ | |align="center"|– | ||
+ | |- | ||
+ | |'''yCCS''' | ||
+ | |align="center"|– | ||
+ | |align="center"|5.7 | ||
+ | |- | ||
+ | |'''5X Rapid Lig. buf.''' | ||
+ | |align="center"|4 | ||
+ | |align="center"|4 | ||
+ | |- | ||
+ | |'''dH<sub>2</sub>O''' | ||
+ | |align="center"|2.7 | ||
+ | |align="center"|2.1 | ||
+ | |- | ||
+ | |'''T4 DNA ligase''' | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |- | ||
+ | | | ||
+ | |align="center"|'''20''' | ||
+ | |align="center"|'''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 | ||
+ | |||
+ | ====ON cultures==== | ||
+ | *pMA.His | ||
+ | *pEX (not yet verified) | ||
+ | *pEX.RFP 3 (not yet verified) | ||
+ | |||
+ | 3 ml LB + 100 Amp. 30 °C ON. |
Revision as of 22:03, 21 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
Assembly of CPP⋅protein⋅His constructs
Continued from 19/8
Transformation results
- 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.
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
ON cultures
- pMA.His
- pEX (not yet verified)
- pEX.RFP 3 (not yet verified)
3 ml LB + 100 Amp. 30 °C ON.