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Lab Meeting Schedule Spring 08 ( at 10.00 in C10:109d) Date Present Coordinator (+snacks) January 18 Roundtable Stefan February 1 Team Building Planning Stefan Events -Mo-Fri 25-29 Feb 2008: PhD course (lots of interesting talks!) -Fri December 14: Christmas Party!

Instrument responsibility

Lab responsibilities

Name	Job	Job frequency
Stefan B	Robocycler PCR (service and checkups)	Continuously
Hannes	Flow cytometer	Continuously
Stefan	Phosphorimager	Continuously
Friederike	Fluorescence microscopes (confocal+upright+inverted)	Continuously
Hannes	dHPLC system (DNA-Bio-LC)	When needed
Stefan	Sequencing robot (MWG Roboseq)	When needed
Xinmei	Chromo4-QPCR	Continuously
Sara	Gel Electrophoresis/Gel Documentation	Weekly
Katrin	Ultracentrifuge	Continuously

Lab week responsibilities

Name	week
Sara	v 1
Xinmei	v2

Protocols and Forms

Cloning and sequencing

PCR with primers M13f and M13r, which target sites on the pCR4-TOPO vector

Master Mix volume volume Final concentration M13f (-20) (20x100 nM) 1µl 1.5 µl 100nM M13r (20x100 nM) 1µl 1.5 µl 100nM PCR buffer 10x 2µl 3 µl 1x (1.5 mM MgCl2) dNTPs (10x200µM) 2µl 3 µl 200µM each dNTP Finnzyme (2 units/µl) 0.2µl 0.3 µl 0.02U Sterile water to 20 µl 12.8µl 19.7 µl DNA extract 1µl 1 µl Total 20 µl 30 µl Finnzyme PCR buffer include 1.5 mM MgCl2

DNA (TOPO guide dilute DNA 10 times, but not Alex) M13f-20 (5’-GTAAAACGACGGCCAG-3’) and M13r (5’-CAGGAAACAGCTATGAC-3’; Invitrogen) PCR condition 95 1min 94 4 min 25 cylces 25 cycles (20 cycle ok!) 95 1 min 94 30 sec 55 1 min 55 30 sec 72 2 min 72 1 min 72 5 min 72 6 min Robot mashine BIO-RAD mashine Check PCR product in 1% gel, choose the clones with right band size (714bp by image gel or 718bp calculated) for RFLP or sequencing For sequencing For RFLP and Sequencing PCR product 30 µl PCR product 30 µl 4 µl for gel check 4 µl for gel check and quantification

                                                                                6 µl for RFLP

26 µl for purification 20 µl for purification To 20 µl to 20 µl 4 µl for quantification 6 µl for quantification 16 µl left 14 µl left 2 µl of 16 for sequencing 3 µl for sequencing?

If don’t extract DNA, pick with toothpick some cells to PCR tubes, run PCR as follow: 94 10 min 25 cycle of: 94 30 sec 55 30 sec 72 30 sec 72 10 min

Prepare sequencing according to guide from UGC

(Run PCR product in 1% gel, right band size PCR product will be chosen for sequencing.) Purifying the PCR products with QiAquick PCR purification kit (not gel purification), quantifying them and preparing sequencing solution as follow:

Sequencing solution Final concentration DNA (10 ng/µl) 2.5µl 25ng (15-30 ng) M13f (2 µM) 2 µl 4 pmoles MQ Water 13.5 µl Total 18 µl