Team:Stockholm/27 July 2010

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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

Jensen et al. Nucleic Acids Res. 2009, 37(Database issue):D412-6

[http://string.embl.de/version_8_3/newstring_cgi/show_network_section.pl?identifiers=9606.ENSP00000331746%250D9606.ENSP00000295600%250D9606.ENSP00000364016%250D9606.ENSP00000371175%250D9606.ENSP00000269280%250D9606.ENSP00000363700%250D9606.ENSP00000360157%250D9606.ENSP00000373571%250D9606.ENSP00000291700&channel1=off&channel2=off&channel3=off&channel4=on&channel5=on&channel6=on&channel7=on&interactive=yes&network_flavor=actions&targetmode=proteins]

[http://string.embl.de/version_8_3/newstring_cgi/show_network_section.pl?identifiers=9606.ENSP00000331746%250D9606.ENSP00000295600%250D9606.ENSP00000226877%250D9606.ENSP00000371175%250D9606.ENSP00000269280%250D9606.ENSP00000363700%250D9606.ENSP00000360157%250D9606.ENSP00000373571%250D9606.ENSP00000291700&channel1=off&channel2=off&channel3=off&channel4=on&channel5=on&channel6=on&channel7=on&additional_network_nodes=5&interactive=yes&network_flavor=actions&targetmode=proteins]

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.

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: