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From 2010.igem.org

Contents 1 Hassan 2 Johan 2.1 PCR 3 Mimmi 3.1 LMWP 3.1.1 glycerol stock + preparation of plasmids 3.1.2 ligation

Hassan

• tyrp1
• nalp1
• mitf
• CGRP
• S100B
• foxd3
• GGA1
• LMP7
• TAPBP

Johan

PCR

The PCR samples that was run on a gel to verify that the colonies had been successfully site-directed mutagenesised seemed to be lost, and a new colony PCR was performed to verify the site-directed mutagenesis before cleaving and adding it to the target vectors.

The ladder is FastRuler DNA ladder low range, but the bands does not show the expected size. Did I mistakenly use the FastRuler DNA ladder middle range?

Mimmi

LMWP

glycerol stock + preparation of plasmids

Mix
LB	12ml
Amp	24µl
ON culture	120µl

• Shake in 37°C, ~200rpm, ~3h

• Add 1600µl culture to pre-made jar with 400µl sterile glycerol

• Take 5ml culture and follow the mini plasmid prep. kit 1 protocol.

• • Wash 2X with EtOH solution

• • Dilute in 50µl HPL6 H₂O

--> in the freezer (plasmid & DNA ) pEX.BBa_J18930A -> 32A

ligation

LMWP_N_F1	2µg/µl
LMWP_CN_F2	1.6µg/µl
LMWP_CN_R1	2µg/µl
LMWP_N_R2	1.5µg/µl
LMWP_C_F1	1.8µg/µl
LMWP_C_R2	1.5µg/µl

Mix	(µl)
F1	1
F2	1
R1	1
R2	1
H2O	4
DNA sol.	42
tot	50µl

• Heatshock 90°C 3min

• Incubate 37°C 1h

Mix		3X
H2O	5
plasmid	2
T4 buffer	1
T4 ligase	1
DNA	1
tot	10µl

• Incubate 22°C 1-3h

• Thaw 3x100µl Top10 competent cells

• Add 1µl plasmid

• Hold on ice 30min

• Heat shock 42°C 50s

• Cool down on ice

• Add 900µl LB

Incubate 37°C 1h 250rpm

• Spin down cells 13000rpm 15s

• Remove 900µl, resuspend in remaining 100µl

• Plate 100µl on LB agar plates with Cm^r

• Grow in 37°C ON