Team:Stockholm/30 August 2010
From 2010.igem.org
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====ON cultures==== | ====ON cultures==== | ||
SH1 and SH2 selected for plasmid prep and sequencing. Set 5 ml LB + 100 Amp ON cultures. 37 °C, 225 rpm. | SH1 and SH2 selected for plasmid prep and sequencing. Set 5 ml LB + 100 Amp ON cultures. 37 °C, 225 rpm. | ||
+ | |||
+ | ===N-CPP extraction=== | ||
+ | ====Gel extraction==== | ||
+ | ''From 28/8 samples'' | ||
+ | Purification using the E.Z.N.A. Gel Extraction kit. Elution in 30 μl dH<sub>2</sub>O; double elution. | ||
+ | |||
+ | {|border="1" cellpadding="1" cellspacing="0" | ||
+ | |+ align="bottom"|† ''Phenolate contaminated sample? High absorbtion at 230 nm'' [http://en.wikipedia.org/wiki/Nucleic_acids_analysis#Other_common_contaminants] | ||
+ | !colspan="3"|DNA concentrations | ||
+ | |- | ||
+ | !Sample | ||
+ | !Conc. [ng/μl] | ||
+ | !A<sub>260</sub>/A<sub>280</sub> | ||
+ | |- | ||
+ | |Tra10 | ||
+ | |align="center"|13.56 | ||
+ | |align="center"|1.86 | ||
+ | |- | ||
+ | |TAT | ||
+ | |align="center"|1.736 | ||
+ | |align="center"|1.20 | ||
+ | |- | ||
+ | |LMWP † | ||
+ | |align="center"|2.523 | ||
+ | |align="center"|2.69 | ||
+ | |} | ||
+ | |||
+ | ====Gel verification==== | ||
+ | [[image:Gelver_extr_CPP_30aug.png|200px|thumb|right|'''Gel verification of extracted N-CPPs.'''<br />3 μl λ; 2 μl sample.<br />λ=GeneRuler 50 bp DNA ladder]] | ||
+ | Ran a gel to verify that the presence and size of our extracted DNA fragments. | ||
+ | |||
+ | 1 % agarose, 100 V | ||
+ | |||
+ | '''Results'''<br /> | ||
+ | Weak band for Tra10, no bands for TAT and LMWP. Proceeded to cloning anyway. | ||
+ | |||
+ | ===Cloning of N-CPPs into pSB1C3=== | ||
+ | A last-minute decision was made to also make a bulk cloning of all three N-CPPs by digesting directly from the N-CPP cluster vector. | ||
+ | ====Digestion==== | ||
+ | [N-CPP plasmid]=672 ng/μl (28/8) | ||
+ | {|border="1" cellpadding="1" cellspacing="1" | ||
+ | |align="center"|[ng/μl] | ||
+ | !Tra10 | ||
+ | !TAT | ||
+ | !LMWP | ||
+ | !N-CPP | ||
+ | |- | ||
+ | |10X FD buffer | ||
+ | |align="center"|3 | ||
+ | |align="center"|3 | ||
+ | |align="center"|3 | ||
+ | |align="center"|3 | ||
+ | |- | ||
+ | |dH<sub>2</sub>O | ||
+ | |align="center"|3 | ||
+ | |align="center"|3 | ||
+ | |align="center"|8 | ||
+ | |align="center"|23 | ||
+ | |- | ||
+ | |FD XbaI | ||
+ | |align="center"|0.5 | ||
+ | |align="center"|0.5 | ||
+ | |align="center"|0.5 | ||
+ | |align="center"|0.5 | ||
+ | |- | ||
+ | |FD AgeI | ||
+ | |align="center"|0.5 | ||
+ | |align="center"|0.5 | ||
+ | |align="center"|0.5 | ||
+ | |align="center"|0.5 | ||
+ | |- | ||
+ | |DNA | ||
+ | |align="center"|23 | ||
+ | |align="center"|23 | ||
+ | |align="center"|18 | ||
+ | |align="center"|3 | ||
+ | |- | ||
+ | | | ||
+ | !30 | ||
+ | !30 | ||
+ | !30 | ||
+ | !30 | ||
+ | |} | ||
+ | |||
+ | Incubation: 37 °C, 30 min<br /> | ||
+ | Inactivation: 80 °C, 10 min | ||
+ | |||
+ | =====Dephosphorylation===== | ||
+ | Treated the N-CPP sample with FastAP alkaline phosphatase to prevent multiple insertions (3, 5, etc...) into target vector. | ||
+ | *3 μl FastAP | ||
+ | **Incubation: 37 °C, 10 min | ||
+ | *Inactivation at 75 °C, 5 min | ||
+ | |||
+ | ====Ligation==== | ||
+ | [Dig. pSB1X3 X+A EXTR]=13.72 ng/μl (digested and extracted vector from 9/8) | ||
+ | [Dig. N-CPP X+A]=60 ng/μl | ||
+ | |||
+ | {|border="1" cellpadding="1" cellspacing="0" | ||
+ | |align="center"|[ng/μl] | ||
+ | !N-CPP | ||
+ | !Tra10 | ||
+ | !TAT | ||
+ | !LMWP | ||
+ | |- | ||
+ | |Vector DNA | ||
+ | |align="center"|3 | ||
+ | |align="center"|3 | ||
+ | |align="center"|3 | ||
+ | |align="center"|3 | ||
+ | |- | ||
+ | |Insert DNA | ||
+ | |align="center"|9 | ||
+ | |align="center"|12 | ||
+ | |align="center"|12 | ||
+ | |align="center"|12 | ||
+ | |- | ||
+ | |5X Rapid Lig. buf. | ||
+ | |align="center"|4 | ||
+ | |align="center"|4 | ||
+ | |align="center"|4 | ||
+ | |align="center"|4 | ||
+ | |- | ||
+ | |dH<sub>2</sub>O | ||
+ | |align="center"|3 | ||
+ | |align="center"|0 | ||
+ | |align="center"|0 | ||
+ | |align="center"|0 | ||
+ | |- | ||
+ | |T4 DNA Ligase | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |- | ||
+ | | | ||
+ | !20 | ||
+ | !20 | ||
+ | !20 | ||
+ | !20 | ||
+ | |} | ||
+ | |||
+ | ====Transformation==== | ||
+ | Standard transformation protocol. | ||
+ | *2 μl ligation mix | ||
+ | *Cm 25 |
Revision as of 12:21, 31 August 2010
Contents |
Andreas
Cloning of SOD into pMA.His
Transformation results
From 28 28/8 Good colony yield. Four colonies (SH1-SH4) picked for colony PCR.
Colony PCR
- SH1-SH4: pMA.SOD⋅His
- PC: Positive control; pMA.His
- NC: Negative control; blank
Procedures according to standard colony PCR protocol. Elongation time 1:00.
Gel verification
1 % agarose, 100 V
Expected bands:
- pMA.SOD⋅His: 831 bp
- pMA.His: 348 bp
Results
Well corresponding bands indicating successful insertion of SOD into the vector.
ON cultures
SH1 and SH2 selected for plasmid prep and sequencing. Set 5 ml LB + 100 Amp ON cultures. 37 °C, 225 rpm.
N-CPP extraction
Gel extraction
From 28/8 samples Purification using the E.Z.N.A. Gel Extraction kit. Elution in 30 μl dH2O; double elution.
DNA concentrations | ||
---|---|---|
Sample | Conc. [ng/μl] | A260/A280 |
Tra10 | 13.56 | 1.86 |
TAT | 1.736 | 1.20 |
LMWP † | 2.523 | 2.69 |
Gel verification
Ran a gel to verify that the presence and size of our extracted DNA fragments.
1 % agarose, 100 V
Results
Weak band for Tra10, no bands for TAT and LMWP. Proceeded to cloning anyway.
Cloning of N-CPPs into pSB1C3
A last-minute decision was made to also make a bulk cloning of all three N-CPPs by digesting directly from the N-CPP cluster vector.
Digestion
[N-CPP plasmid]=672 ng/μl (28/8)
[ng/μl] | Tra10 | TAT | LMWP | N-CPP |
---|---|---|---|---|
10X FD buffer | 3 | 3 | 3 | 3 |
dH2O | 3 | 3 | 8 | 23 |
FD XbaI | 0.5 | 0.5 | 0.5 | 0.5 |
FD AgeI | 0.5 | 0.5 | 0.5 | 0.5 |
DNA | 23 | 23 | 18 | 3 |
30 | 30 | 30 | 30 |
Incubation: 37 °C, 30 min
Inactivation: 80 °C, 10 min
Dephosphorylation
Treated the N-CPP sample with FastAP alkaline phosphatase to prevent multiple insertions (3, 5, etc...) into target vector.
- 3 μl FastAP
- Incubation: 37 °C, 10 min
- Inactivation at 75 °C, 5 min
Ligation
[Dig. pSB1X3 X+A EXTR]=13.72 ng/μl (digested and extracted vector from 9/8) [Dig. N-CPP X+A]=60 ng/μl
[ng/μl] | N-CPP | Tra10 | TAT | LMWP |
---|---|---|---|---|
Vector DNA | 3 | 3 | 3 | 3 |
Insert DNA | 9 | 12 | 12 | 12 |
5X Rapid Lig. buf. | 4 | 4 | 4 | 4 |
dH2O | 3 | 0 | 0 | 0 |
T4 DNA Ligase | 1 | 1 | 1 | 1 |
20 | 20 | 20 | 20 |
Transformation
Standard transformation protocol.
- 2 μl ligation mix
- Cm 25