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From 2010.igem.org

Contents 1 Johan 1.1 Colony PCR 2 Nina 2.1 Transformation of IgG protease in shipping vector 2.2 Mini prep of CPP transportan 10 version C 3 Mimmi 3.1 MITF 3.1.1 colony PCR 3.1.2 Culture

Johan

Colony PCR

• A colony PCR on the TAT CCP (N-version) was performed as the result from the last gel electrophoresis (insert date) was just a smear on almost all bands. The reason was most probably too much genomic DNA (too much colony picked), so less of the colonies was picked and the final volume was increased from 25 µl to 50 µl.

• A colony PCR on the LMWP CCP (N- & C-version) was performed as the result from Mimmis gel electrophoresis was just empty bands (insert date).

• TAT: colonies #1-41 +insert, #42-43 -insert.
• LMWP: colonies #1 -insert, #3-24 +insert.

• PCR reaction mix
  • 1 µl Pfu polymerase
  • 1 µl 10 mM dNTPs
  • 3 µl 10 µm f.primer (VF2)
  • 3 µl 10 µm r.primer (VR)
  • 5 µl Pfu buffer 10x
  • 32 µl H2O (miscalculation, should have been 37 µl)

• PCR program
  • 98 °C - 2 min
  • 35 cycles of
    • 98 °C - 10 sec
    • 55 °C - 15 sec
    • 72 °C - 30 sec
  • 72 °C - 5 min
  • 4°C - ∞

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Nina

Transformation of IgG protease in shipping vector

I transformed 100 ul of competent top 10 cells with 3 ul IgG protease in the shipping vector. This was plated on dishes with chloramphenicol resistance.

The transformation method was carried out according to the procedure described in protocols.

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Mini prep of CPP transportan 10 version C

I carried out a mini prep of CPP transportan 10 version C colony numbers 9 and 40.

The mini prep method was carried out according to the procedure described in protocols.

Measuring concentration with spectrophotometer:

Mimmi

MITF

colony PCR

Mix	(µl)	X8		F primer: pSB1_VF2		conditions
H2O	22.5	180		R primer: pSB1_VR		time	°C
F primer	1	8				2m	94
R primer	1	8				30s	94	)
DNA	0.5	8X0.5				30s	60	> 30 cycles
tot	25µl					45s	72	)
						10m	72
						oo	10

Culture

Mix
LB	5ml			Gel
Cm	10µl		well	sample
Colony	9.5µl		1	ladder
			2	CA
			3	CB
			4	CC
			5	NA
			6	NB
			7	NC
			8	control
			9	blank control

• Something wrong in the PCR conditions?

--> Try longer elongation time

--> Mix more carefully...

• Give to Nina and Johan
  • pSB1T3.SOD
  • 3 plates of Cm
  • 3 plates of Km
  • colonies with LMWP
  • plates with LMWP