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From 2010.igem.org

Contents 1 Mimmi 1.1 over-expression in pEX 1.2 Site-Directed Mutagenesis 1.2.1 Primers 1.2.2 reaction 1.3 Transformation 2 Hassan 3 Nina 4 Colony PCR on IgG protease in shipping vector 5 Mini prep on IgG protease and CPP 6 Sequencing CPP TAT N version

Mimmi

over-expression in pEX

- remaking the comassie-gel

Mix	v
LB	4 ml
glycose	40 µl (1%)
Amp	8 µl (0.5%)
old culture	40 µl

• Grow in 37C, ~200 rpm ~2h until OD = 0.6

• Add IPTG (1 µl/ml,1M)in one of the two cultures, the other one is used as a control

• Take sample at 0h, 1h, 2h, 3h
  • pipette 500µl into a 1.5ml tube
  • spinn down the sells and remove LB
  • resuspend in 50µl loading dye
  • freeze

Site-Directed Mutagenesis

Primers

(MW/10)/(OD*33) = dilution factor

1000/df = volyme to get 100µM primer

100µM = (MW/10) ng/µl

(MW/10)/125 = df to get 125 ng/µl

primer	V H2O (µl)	V H2O to get 125 ng/µl (µl)
MITF_site1_F	463.88	3µl + 15.4µl
MITF_site1_R	64.77	3µl + 15.3µl
MITF_site2_F	144.85	3µl + 21µl
MITF_site2_R	141.77	3µl + 21.6µl
	concentration
yCCS_F	1085.7 ng/µl	3µl + 23µl
yCCS_R	996.5 ng/µl	3µl + 20.9µl

reaction

Mix	(µl)	X2
H2O	40	80
dNTPs	1	2
F primer	1	2
R primer	1	2
DNA	1	2*1
Pfu X10 buffer	5	10
Pfu turbo pol	1	2

MITF	yCCS	conditions
MITF_Site1_F	yCCS_F	time	°C
MITF_Site2_R	yCCS_R	30s	95
pRc/CMV	pSB1C3	30s	95
~6.7kb	~3,5kb	30s	55
		7m	68
		oo	4

• Make sure sample is ≤ 37°C
• Add 1µl Dpn1 (to 50µl product) and incubate in 37°C ON

Transformation

mix

Top 10 competent cells 100µl

pRc/CMV.MITF_M 1µl

• Hold on ice 30min
• Heat shock 42&degC, 55sec
• Cool down 1min (on ice)
• Add 900µl LB
• Incubate 37°C, 250rpm, 1h (forgot rpm)
• Spinn down cells, 13000rpm, 15sec
• Remove 900µl LB
• Plate the 100µl on a Amp-plate
• Grow ON at 37°C

Hassan

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[1]

[2]

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Nina

Colony PCR on IgG protease in shipping vector

I made a colony PCR on the IgG protease that I have inserted into the iGEM shipping vector to verify that the gene is inserted in a correct position. Therefore I used one of the gene's primers and one of the vector's verification primers. The colony numbers are: 1, 2, 3, 4 and 5.

PCR reaction mix:

• 1 µl Morten's polymerase PjuX7

• 1 µl 10 mM dNTPs

• 3 µl 5 µM forward primer (VF2)

• 3 µl 5 µM revers primer (gene's primer)

• 10 µl buffer 5X

• 1 µl MgCl2 50mM

• 30 µl H2O

• DNA template was one colony

PCR program:

98°C - 2 min

31 cycles of:

• 98°C - 10 sec

• 55°C - 15 sec

• 72°C - 1.5 min

72°C - 5 min

4°C - ∞

DNA Ladder: FastRuler™ Middle Range, ready-to-use, 100-5000 bp Fermentas

Colony number 2 looks good on the gel. This one will be inoculated in LB in order to become minipreped to be shipped to iGEM hq.

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Mini prep on IgG protease and CPP

I prepare a miniprep on the inoculated IgG protease with colony number 2. In addition I miniprep three inoculated samples with vector carrying CPP from colony number 22, 23 and 33.

The method is carried out according to the procedure in protocols.

Measuring concentration with spectrophotometer:

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Sequencing CPP TAT N version

I send three samples of CPP TAT N version for sequencing. Colony numbers are: 22, 23 and 33.

• 15 ul vector

• 1.5 ul 10uM VR2 primer

22: ASB0045 105

23: ASB0045 104

33: ASB0045 103