Cloning and sequencing

Prepare before start
•	LB liquid medium

•	Kanamycin-need to keep dry, stock 10 mg/ml in water, storage -20 degree and working concentration 50 µg/ml, it needs to dilute 200 times from 10 mg/ml 10 mg/ml : 50 mg to 5 ml MQ sterile water – filtrating through a 0.22-µm filter

•	Steile liquid LB add kanamycin to 50 µg/ml

•	Solid LB medium, autoclave and add kanamycin when temperature drop down to 40 to 50 degree, and pool medium to plate(20 ml per plate), keep dark in fridge for 1 month

•	X-gal: 4% (Alex) w/v in dimethylformamide (2% in molecular book) 40 mg/ml storeage at -20 degree wrapping in aluminum foil to avoid light 40 µl (=1.6 mg/plate) spread into one plate Should prepare 2%, it may work with less than 40 µl

•	14% DMSO (liquid) in LB 1.4 ml DMSO to 8.6 ml LB liquid, doesn’t need to autoclave, store in fridge

Normal PCR with DyNAzyme II
PCR buffer 10x                   2µl

A344f 20x250nM                   1µl

A915r 20x250nM                   1µl

dNTP 10x200 µM                   2µl

MgCL2 (25 mM)                    0.8 µl             1 mM

Water                            12µl

DyNAzyme II (2U/µl)              0.2µl (0.02U/µl)


 * DNA                            1µl (10 fold diluted with buffer EB)

Total                            20µl

Total 2.5 mM MgCl2 better amplification than 1.5 mM MgCl2

PCR reaction:

94  3 min

32 cycle of (depend on DNA concentration)

94  30 sec

61  30 sec

72  30 sec

72  7 min

Check with 1% gel to see how the PCR product.

Real-time PCR can’t be used for cloning, because Answer from Kaisa Helminen about SYBR Green: (Thank you for contacting Finnzymes. As our DyNAmo master mixes include dUTP, UNG negative strain must be used for transforming the cloned product. Otherwise UNG activity will break the inserted fragment. An example of cloning dUTP containing products is found in the following web site: http://www.retroconference.org/2003/cd/Abstract/580.htm We do not have information about how TOPO and other system tolerate SYBR green dye, but if needed, SYBR green can be removed by simple ethanol precipitation.)

Gel purification of normal PCR products
1) (I directly load the PCR product in gel and purify the gel. Use the wide comb, 30 µl can be loaded, 20 µlx3=60 µl, two lines)

Using QiAquick gel extraction kit (Qiagen) and quantification by low mass DNA ladder.

Loading 30µl DNA in 1% agarose gel (I prepared 70 ml of 1% gel with 7 ml of EtrB. Pour in small gel tray with wide and thick comb. Loading 30 µl samples with 3 µl of dye, running for 40 min. 6µl of 100bp as ladder) (Fig.1). Excise the DNA fragment from the agarose gel with a sharp, clean razor. Weigh gel slice and add three volumes of QG buffer to one volume of gel (100 mg~100µl, mxium 400 mg in one column). Follow instruction from the QIAquick Spin handbook using microcentrifuge protocol. Concentrated to 30 µl buffer EB. Quantify the DNA concentration with 1% gel loading 6µl samples with 1 µl dye, 120 V, 85mA and 40 min. First run with SYBR PCR product Fig.1 30 µl DNA in gel with 100 bp ladder Fig. 2 Quantification after gel purification with 6 µl with Low mass DNA ladder (Lane 1 RK1 38.5 ng/µl, lane 2 1-77, 35.2 ng/µl and lane 3 1-77 7min, 27.1 ng/µl)

Performing the TOPO cloning reaction (refer TOPO TA Cloning Kit for sequencing)
Prepare:

•	Take out vector, water and salt solution 10 min before mixing

Fresh PCR product (1-2 days old)	? DNA should be 5-10 ng Salt solution	                       1 µl Water	                               ? µl TOPO vector	                       1 µl Total	                               6 µl

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.

Prepare:

•	15 min after mixing vector, taking competent cell out from -80 freezer, melting in ice,

•	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)

Transformation One Shot TOP10 competent cells
Procedure (2 library need: 1 vial of competent cells and 4 LB plates, if using half kit) One tube to 2 library:

3 µl (because I only use 0.5 µl vector half the protocol says) of TOPO Cloning reaction

25 µl of One Shot Chemically competent E. coli (50 µl, Alex has used 50 µl for 2 reactions). One tube to 1 library:

2.5 µl of TOPO Cloning reaction for old kit

50 µl of one Shot Chemically competent E. coli

•	Mix gently (flap the tube by hand softly, turn around the tubes a little bit). Important!

•	Incubate on ice for 30 min (5 to 30 min) (longer time has no effect on efficiency).

Prepare: •	After incubating, taking out S.O.C medium to room temperature

•	20 min after incubation, switch on 42 degree waterbath (10 min is enough for warming up)

•	Heat-shock the cells for 30 seconds at 42 degree without shaking (I count from 1 to 35 for 30 seconds)

•	Immediately transfer the tubes to ice

•	Add 250 µl of room temperature S.O.C medium

•	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

Prepare

•	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

•	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

•	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.

•	Colones from each plate?

RK2	Old kit	New kit

50 µl	63 (total)	666 (total)

150 µl	244 (total)	Uncountable (total)

Incubation start at 7 PM Yesterday, check in the morning today at 9:30 AM, there is almost no colonies Grow transformants in liquid LB with 50 µg/ml kanamycin for PCR with M13 primer, RFLP or T-RFLP and sequencing

(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.)

•	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.

•	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.

Prefer colonies with sufficient distance from neighbors over those in aggregates.?

•	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.

•	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).

DNA extraction from clones
•	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

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?

Check PCR product in 1% gel, choose the clones with right band size (714bp by image gel or 718bp calculated) for RFLP or 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 DNA 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