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	*0.5% agarose gel		*0.5% agarose gel
	*(Melted in our bath, redoing the gel in another bath...		*(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.
		+
		+	{{Stockholm/Footer}}

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Latest revision as of 10:46, 26 October 2010

Contents 1 Andreas 1.1 Plasmid prep. 1.2 Cloning 1.2.1 Digestion 1.2.2 Ligation 1.2.3 Quick transformation 2 Mimmi 2.1 MITF 2.1.1 - restriction enzyme 2.1.2 - Gel 3 Nina 3.1 Overnight culture of Tyrosinase

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.

1. Gentle mixing
2. Incubation in 37 °C, 15 min
3. Tubes returned to ice
4. 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

1. Gently mixing
2. Incubation 22 °C, 10 min
3. Collection by 5 sec centrifugation
4. Tubes returned to ice

Quick transformation

1. 100 μl Top10 cells thawed
2. 2 μl ligation mixture added to cells. Cells kept on ice ≈5 min
3. Heat-shock in 42 °C, 30 sec
4. Cells plated on LB agar with relevant antibiotics
   • 100 Amp
   • 25 Cm
5. 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.