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| '''[[Cloning and sequencing]]''' | | '''[[Cloning and sequencing]]''' |
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| == Performing the TOPO cloning reaction (refer TOPO TA Cloning Kit for sequencing) ==
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| Prepare:
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| • Take out vector, water and salt solution 10 min before mixing
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| Fresh PCR product (1-2 days old) ? DNA should be 5-10 ng
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| Salt solution 1 µl
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| Water ? µl
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| TOPO vector 1 µl
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| Total 6 µl
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| Mix reaction gently (flap by finger a little bit) and incubate for 30 min (5-30 min) at room temperature (22-23 degree) (page 5 in manual). Place the reaction on ice and proceed to transforming.
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| Prepare:
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| • 15 min after mixing vector, taking competent cell out from -80 freezer, melting in ice,
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| • 5 min before transformation, taking gently 25 µl competent cell to a round bottom tube (easy to mix now, even later after adding 250 µl S.O.C) by using tip-cut 200 µl tips. (my first trial failed)
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| Transformation One Shot TOP10 competent cells (page 9)
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| Procedure (2 library need: 1 vial of competent cells and 4 LB plates, if using half kit)
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| One tube to 2 library:
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| 3 µl (because I only use 0.5 µl vector half the protocol says) of TOPO Cloning reaction
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| 25 µl of One Shot Chemically competent E. coli (50 µl, Alex has used 50 µl for 2 reactions).
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| One tube to 1 library:
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| 2.5 µl of TOPO Cloning reaction for old kit
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| 50 µl of one Shot Chemically competent E. coli
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| • Mix gently (flap the tube by hand softly, turn around the tubes a little bit). Important!
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| • Incubate on ice for 30 min (5 to 30 min) (longer time has no effect on efficiency).
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| Prepare:
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| • After incubating, taking out S.O.C medium to room temperature
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| • 20 min after incubation, switch on 42 degree waterbath (10 min is enough for warming up)
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| • Heat-shock the cells for 30 seconds at 42 degree without shaking (I count from 1 to 35 for 30 seconds)
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| • Immediately transfer the tubes to ice
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| • Add 250 µl of room temperature S.O.C medium
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| • Cap the tube tightly and shake the tube horizontally (200 rpm, I use 160 rpm because there is no 200 rpm in the control, or 160 or 240) at 37 degree for 1 hour
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| Prepare
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| • Take out LB plates to warm up at 37 degree, after 30 min, spread X-gal on plates and put them back to 37 degree until use
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| • Spread 10-50 µl (I spead one plate with 50 µl + 50 µl S.O.C medium and one with 150 µl without S.O.C medium for the old kit. New kit, spread one LB plate with 20 µl (+80 µl S.O.C medium) and one LB plate with 50 µl (+50 µl S.O.C medium) are enough) on X-gal and warm LB plates
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| • Each transformation on a pre-warmed selective plate (with X-gal) and incubate overnight (need 24 h, you can distinguish different colour colonies) at 37 degree.
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| • Colones from each plate?
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| RK2 Old kit New kit
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| 50 µl 63 (total) 666 (total)
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| 150 µl 244 (total) Uncountable (total)
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| Incubation start at 7 PM Yesterday, check in the morning today at 9:30 AM, there is almost no colonies
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| Grow transformants in liquid LB with 50 µg/ml kanamycin for PCR with M13 primer, RFLP or T-RFLP and sequencing
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| (I didn’t add X-gal in the first, there will be no colour. According to the TOPO protocol, all clones grown should be positive, but Stefan said it was not 100% true.)
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| • Select at least 15 positive clones per library (per reactor) with autoclaved toothpicks into the wells of clear microtiter plates (96 wells) (NUNC cell culture plates, sterile) containing 150 µl LB broth with 50 (100 ? Alex use 50 µg) µg/ml kanamycin.
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| • Incubate overnight (approx 24 h) at 37 degree with 200 rpm agitation (160 rpm I used) on a moisture box. Check the growth toward light.
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| Prefer colonies with sufficient distance from neighbors over those in aggregates.?
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| • After growth, 75 µl from each well should be transferred to a PCR 96-well plates using multi channel-pipettor. Store these PCR plates (why PCR plates, because DNA extraction needs to heat 98 degree for 10 min in PCR mashine) immediately at -80 until DNA extraction for PCR and sequencing or immediately run DNA extraction.
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| • The Original microtiter 96-well plates: add 75 µl of 14% DMSO in LB broth (*Dimethyl Sulfoxide 二甲基亚砜 (CH3)2SO, DMSO also sees use as a cryoprotectant, added to cell media in order to prevent the cells dying as they are frozen. Approximately 10% may be used with a slow-freeze method, and the cells may be frozen at -20°C or stored in liquid nitrogen safely*) into each well (7% of DMSO in the final solution), and then keep at -80 (-20?) for long term storage with cells can grow later (clone library).
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Lab Meeting Schedule Spring 08 ( at 10.00 in C10:109d)
| Date
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Present
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Coordinator (+snacks)
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| January 18
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Roundtable
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Stefan
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| February 1
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Team Building Planning
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Stefan
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Events
-Mo-Fri 25-29 Feb 2008: PhD course (lots of interesting talks!)
-Fri December 14: Christmas Party!
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Instrument responsibility
Lab responsibilities
| Name
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Job
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Job frequency
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| Stefan B
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Robocycler PCR (service and checkups)
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Continuously
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| Hannes
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Flow cytometer
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Continuously
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| Stefan
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Phosphorimager
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Continuously
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| Friederike
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Fluorescence microscopes (confocal+upright+inverted)
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Continuously
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| Hannes
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dHPLC system (DNA-Bio-LC)
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When needed
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| Stefan
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Sequencing robot (MWG Roboseq)
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When needed
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| Xinmei
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Chromo4-QPCR
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Continuously
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| Sara
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Gel Electrophoresis/Gel Documentation
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Weekly
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| Katrin
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Ultracentrifuge
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Continuously
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Lab week responsibilities
| Name
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week
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| Sara
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v 1
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| Xinmei
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v2
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Protocols and Forms
Cloning and sequencing
• Centrifuge the PCR plates at maximum speed (4000rpm) for 30 min using microtiter centrifuge.
• Up-side-down in handduk tissues placed on Alumium in sterile hood (Liquid difficult come out, may be Alex’s method is good: Centrifuge up-side-down with paper at 500 rpm for 6 min). Change a new paper until it is dry.
• Add 30 µl of MQ, hold the 96-wells plate and votex
• Heat at 98 degrees for 10 min (in PCR mashine), votex again
• 1µl of DNA use for PCR, the rest PCR product were kept at -80 for several weeks, if it need to repeat the sequencing again.
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
