"

From 2010.igem.org

Contents 1 Andreas 1.1 Cloning results 1.2 ON cultures 1.3 Cloning/transformations 1.4 CPP DNA synthesis 1.5 Fermentas/ThermoFisher Scientific sponsorship 2 Mimmi 2.1 MITF 2.1.1 - Gel 2.1.2 - amplifying and moving

Andreas

Cloning results

From 3/8 transformations

pSB1A3.SOD:	Bacterial lawn on all three plates, probably due to the Amp in plates having degraded. Plates discarded
pSB1A3.yCCS A:
pSB1A3.yCCS B:
pSB1C3.IgG prot.:	Good colony yield. Left to grow and mature for 4-5 more hours until red colonies appeared.

ON cultures

Four colonies picked from IgG protease plate and resuspended in 10 μl LB. 3 μl used to inoculate 5 ml LB + 25 Cm. Grown ON in 37 °C, 250 rpm.

Cloning/transformations

New quick-transformations were performed for:

• pSB1A3.SOD
• pSB1A3.yCCS A
• pSB1A3.yCCS B

Procedures as described 3/8.

CPP DNA synthesis

Since our previous CPP synthesis order was rejected due to repetetive sequences Johan and I redesigned the order into two separate clusters, CPP_N and CPP_C. The new clusters were designed with both restriction sites and primer annealing sites flanking each CPP. An extension sequence was also added to enable gel separation after digestion, as the CPPs are very similar in length.

Johan also redesigned the coding sequence by shifting codons by the addition of "silent mutations".

Fermentas/ThermoFisher Scientific sponsorship

Called Fermentas/ThermoFisher Scientific and got a small (but much appreciated) sponsorship for FastDigest AgeI restriction enzymes. Their logotype was added to our dedicated sponsorship page.

Mimmi

MITF

- Gel

• 0.5% agarose gel
• No products!

--> Primers are double and triple checked... Trying a gradient!

- amplifying and moving

• 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)

dNTP 10µM	(µl)
ATP 100µM	7
CTP 100µM	7
GTP 100µM	7
TTP 100µM	7
sH2O	52	tot	70

Mix	(µl)	X18		conditions
sH2O	39.5	711		time	°C
F primer	0.75	13.5		2m	94
R primer	0.75	13.5		30s	94	)
buffer	5	90		30s	45, 50, 55	> 5 cycles
dNTPs 10µM	1.5	27		1m30s	68	)
MgSO4 50µM	1	18		30s	94	\
polymerase	0.5	6		1m30s	68	/ 25 cycles
DNA	1	18X1		10m	68
tot	50			oo	10