Team:Stockholm/3 August 2010
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I inoculated Tyrosinase in its original vector from a glycerol stock (that was sent to us from iGEM hq) in 12 ml LB and added 24 ul amphicillin (50 mg/ml). This was incubated in 37 °C overnight in shake. This will be mini prepped tomorrow. | I inoculated Tyrosinase in its original vector from a glycerol stock (that was sent to us from iGEM hq) in 12 ml LB and added 24 ul amphicillin (50 mg/ml). This was incubated in 37 °C overnight in shake. This will be mini prepped tomorrow. | ||
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Latest revision as of 10:46, 26 October 2010
Contents |
Andreas
Plasmid prep.
From 2/8 ON cultures
Elution volume: 70 μl.
DNA concentration | ||
---|---|---|
Sample | Conc. [ng/μl] | A260/A280 |
pSB1A3 1 | 87.00 | 1.98 |
pSB1A3 2 | 120.3 | 1.92 |
pSB1A3 3 | 145.7 | 1.94 |
pSB1A3 4 | 58.72 | 2.01 |
pSB1C3 1 | 188.9 | 1.97 |
pSB1C3 2 | 101.2 | 2.00 |
pSB1C3 3 | 201.2 | 1.93 |
pSB1C3 4 | 147.7 | 1.92 |
Cloning
To remove unwanted insertion of a C nucleotide located at the PstI site in the suffix, caused by cloning in the pEX vector, yCCS, SOD and IgG protease will be transferred to a new vector by digestion with SpeI.
Digestion
- IgG protease (on pSB1A3)
- yCCS A (on pSB1C3)
- yCCS B (on pSB1C3)
- SOD (on pSB1C3)
- pSB1C3 2
- pSB1A3 2
Digestion tubes | ||||||
---|---|---|---|---|---|---|
[μl] | IgGp [500] | SOD [120.6] | yCCS A [94.8] | yCCS B [85.8] | pSB1A3 [120.3] | pSB1C3 [101.2] |
10X FastDigest buffer | 5 | 5 | 5 | 5 | 5 | 5 |
dH2O | 39 | 26 | 22 | 20 | 26 | 23 |
DNA (2 μg) | 4 | 17 | 21 | 23 | 17 | 20 |
FD SpeI† | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
FD EcoRI | 1 | 1 | 1 | 1 | 1 | 1 |
50 | 50 | 50 | 50 | 50 | 50 |
† Only 0.5 μl of FD SpeI to save enzyme, since it is very expensive.
- Gentle mixing
- Incubation in 37 °C, 15 min
- Tubes returned to ice
- Promptly proceeded to ligation, without enzyme inactivation or DNA purification.
Ligation
- pSB1C3 + IgG prot.
- pSB1A3 + yCCS A
- pSB1A3 + yCCS B
- pSB1A3 + SOD
Ligation tubes (vector:insert 1:5) | ||||
---|---|---|---|---|
[μl] | IgG + pSB1C3 | SOD + pSB1A3 | yCCS A + pSB1A3 | yCCS B + pSB1A3 |
Vector DNA [100 ng] | 2.5 | 2.5 | 2.5 | 2.5 |
Insert DNA [500 ng] | 12.5 | 12.5 | 12.5 | 12.5 |
5X Rapid Ligation buffer | 4 | 4 | 4 | 4 |
T4 DNA ligase | 1 | 1 | 1 | 1 |
20 | 20 | 20 | 20 |
- Gently mixing
- Incubation 22 °C, 10 min
- Collection by 5 sec centrifugation
- Tubes returned to ice
Quick transformation
- 100 μl Top10 cells thawed
- 2 μl ligation mixture added to cells. Cells kept on ice ≈5 min
- Heat-shock in 42 °C, 30 sec
- Cells plated on LB agar with relevant antibiotics
- 100 Amp
- 25 Cm
- Incubation ON in 37 °C
Mimmi
MITF
- restriction enzyme
- the PCR products (from 2010-08-02) are treated with the restriction enzyme AgeI
- the mutated MITF should show two bands: ~1300bp and ~10bp (which you dont see)
- the control (non-mutated) MITF should show three bands: ~1060bp, ~200bp and ~10bp (which you don't see)
Mix | (µl) |
---|---|
DNA | 15 |
sH2O | 2 |
10X buffer | 2 |
AgeI | 1 |
tot | 20 |
- Mix and spinn down breafly
- Incubate 2h (1-16h)
- Gel
- 0.5% agarose gel
- (Melted in our bath, redoing the gel in another bath...
Nina
Overnight culture of Tyrosinase
I inoculated Tyrosinase in its original vector from a glycerol stock (that was sent to us from iGEM hq) in 12 ml LB and added 24 ul amphicillin (50 mg/ml). This was incubated in 37 °C overnight in shake. This will be mini prepped tomorrow.
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