Cloning

HOW TO ... Clone with the promega PGEM-T kit

Rutgers, November 2004

Media and plates
1.1- Equipment
 * gas burner
 * rotating plate
 * aluminium rack for PCR tubes

1.2- Reagents
 * Tryptone: Fisher BP1421-500
 * yeast extract: Fisher BP1422-500
 * NaCl: Sigma S-3014
 * MgCl2: ????
 * glucose: ???
 * KCl: ???
 * Agar: DIFCO 214010
 * DMSO: ????
 * Ampicillin: Sigma A-9518
 * Xgal (5-bromo--4-chloro-3-indolyl-ß-D-galactopyranoside): Sigma B4252-1G
 * PGEM kit: Promega

1.3- Other consumables
 * gloves
 * sterile (???) plates
 * pH paper
 * pasteur pipettes
 * DW

1.4- LB (Luria-Bertami) agar plates - Tryptone: 5.0 g	- yeast extract: 2.5 g	- NaCl: 5.0 g	- Ampicillin: dissolve 50 mg in about 100 µl DW	- Xgal: dissolve 50 mg in about 250 µl DMSO
 * Mix in DW:
 * Stir well on mag stirrer
 * Once dissolved, add Agar: 7.5 g
 * Add about 4 drops 10 N NaOH, check with pH-paper that pH is 7.0. Adjust if needed.
 * Autoclave @ 121°C for 30 min (liquid setting)
 * Cool until one can pick up the flask without wearing gloves (about 55°C)
 * Prepare additives:
 * Add ampicillin and Xgal, gently swirl
 * Pour the solution on sterile (???) plates
 * Let solidify about 1 h, wrap, label (name, date, additive)
 * keep at 4°C

1.5- IPTG stock solution (100 nM)
 * Dissolve 238 mg IPTG (sigma I-6758) in 10 ml DW
 * store in 1 ml aliquotes at -20°C

1.6- SOC medium for 1 l			500 ml		100 ml	tryptone			20 g			10 g			2 g	yeast				5 g				2.5 g			0.5 g	NaCl				0.5 g			0.25 g			0.05 g	250 nM KCl		10 ml			5 ml			1 ml						for 1 l			500 ml		100 ml	ddH2O(XXX)		980 ml		490 ml		98 ml						for 1 l			500 ml		100 ml	1 M MgCl2		10 ml			5 ml			1 ml						for 1 l			500 ml		100 ml	2 M glucose		10 ml			5 ml			1 ml
 * 1 M MgCl2 stock: dissolve 20.33 g MgCl2 6H2O in 100 ml ddH2O (XXX), autoclave on liquid cycle @ XXX°C for 20 min (can be done at the same time as SOC pre-mix below)
 * 2M glucose stock: dissolve 18 g glucose into 50 ml (final volume) ddH2O (xxx) and filter-sterilize into sterile 50 ml tube
 * 250 mM KCl stock: dissolve 1.86 KCl in 100 ml ddH2O (XXX)
 * combine:
 * adjust pH to 7.0 w/ NaOH
 * bring to volume:
 * autoclave on liquid cycle @ XXX°C for 20 min
 * add autoclaved 1 M MgCl2
 * add sterilized 2 M glucose
 * aliquote in 2 ml tubes
 * store at -20°C

1.7- DYT medium 500 ml		100 ml	tryptone			8.0 g			1.6 g	yeast				5.0 g			1.0 g 	NaCl				2.5 g			0.5 g
 * combine:
 * mix in DW
 * add 10 N NaOH (about 2.5 drops for 100 ml)
 * check that pH=7.0 with pH paper; adjust as needed
 * aliquote: add 3 ml to 15 ml glass tubes, cap loosely
 * autoclave in a rack in a tub with 4 cm of water
 * store at 4°C

PCR products cloning
The PCR product may require purification first. Use "GenElute agarose spin column" (sigma 5-6500) if the product is in a gel or "QIAquick PCR purification kit" if it is liquid.

2.1- Equipment
 * heating block
 * spinning centrifuge

2.2- Reagents
 * Vector: Promega pGEM-T cloning kit
 * Competent cells: XL2-Blue (Stratagene 200150)
 * SOC medium
 * ß-mercaptoethanol (ß-MAE)

2.3- Other consumables
 * 0.3 µl sterile ??? tubes
 * DW

2.4- Ligation DW		Tp2x	pGEMT	Ligase - for 1 tube: 		1.5 	2.5		0.25		0.5		(total: 4.75) - for 4 tubes:		6.0		10.0	1.0			2.0		(total: 19.0) - for 6 tubes:		9.0		15.0	1.5			3.0		(total: 28.5) - for 8 tubes:		12.0	20.0	2.0			4.0		(total: 38.0) - for 10 tubes:	15.0	25.0	2.5			3.0		(total: 47.5) - for 12 tubes:	18.0	30.0	3.0			6.0		(total: 57.0)
 * Prepare a master-mix in a 0.5 ml tube containing (µl):
 * dispense 4.75 l of the mix into 0.2 ml tubes
 * Add 0.25 µl of the purified PCR-product
 * vortex
 * incubate 1 night at 4°C or 1 h at RT
 * spin before use

2.5- Transformation (IMPORTANT: keep the cc on ice as much as possible)
 * set a heating block at 42°C and fill wells with DW
 * defrost a 2 ml tube of SOC medium
 * place the required LB plates at 37°C
 * set 15 1.5 ml tubes in a recipient full of watery ice
 * cut the head of a yellow tip with a razor blade to increase its diameter
 * take a tube of XL2-blue competent cells (cc) out of the -80°C freezer, put it immediately in watery ice and wait until it thaws
 * gently stir the cc with the pipette tip, slowly resuspend them
 * distribute 10 µl of cc into 10 of the 15 tubes
 * keep the nulber of tubes needed and put the extra ones at -80°C
 * add 0.18 µl of ß-MAE to each tube
 * incubate on watery ice for 10 min, gently shaking with a finger tip 2-3 times during the 10 min
 * add 1.2 µl of each ligation product into each cc-tubes
 * gently mix
 * incubate on watery ice for 30 min
 * heat shock exactly 30 sec at 42°C in the heating block (no mix, no shake)
 * put on ice during 2 min
 * spin
 * add 125 µl of SOC medium to each tube
 * shake the tubes tilted @ 37°C for 1 h at 225 rpm

- prepare the appropriate number of Pasteur pipettes close tip and bend - clean bench using EtOH
 * during this hour, finish preparing the LB plates (stored @ 37°C):
 * when the incubation is complete, spread 145 µl of SOC-CC onto plate
 * let soak 10 min @ 37°C
 * incubate inverted @ 37°C overnight
 * shift to 4°C for 1 h for color development before screening
 * seal the plates with parafilm and store them at 4°C

2.6- Screening 96°C: pause 96°C: 30 sec 94°C: 30 sec 52°C: 30 sec 68°C: 2 min 68°C: 5 min 4°C: pause
 * set up PCR reaction (25 µl) for 3-6 colonies per plate
 * primers are the vector primers m13rev and T7/M13
 * Red TAQ
 * just touch a colony with a sterile toothpick and twirl in PCR tube
 * PCR program (pPCR-JU) has 25 cycles:
 * seal the plates with parafilm and store them at 4°C
 * check how_to_culture_bacteria.txt if the a liquid culture of the bacteria is needed

Cleaning of PCR product
3.1- Equipment
 * material for gel electrophoresis
 * PCR machine

3.2- Reagents
 * Shrimp alkaline phosphatase (SAP), stock solution 1 U/µl [removes P from 3' end of DNA]
 * exonuclease I, stock solution 10 U/µl [removes single strand DNA]

3.3- Other consumables

3.4- Cleaning lid temp: 110.0°C 37.0°C: pause 37°C: 30 min (incubate) 80°C: 15 min (denaturate) 25°C: pause
 * visualize the PCR product to make sure that there is no sub-band (concentration should be 20 ng/ml)
 * add 5 µl of each PCR product into a 200 µl strip tube
 * mix enough SAP and EXO in equal volumes
 * mix and spin
 * distribute 1 µl of this mixture to the lid of each tube
 * Use a PCR machine to incubate and denaturate (SWATI-PURE):
 * The products are then ready for the sequencing reaction and can be stored at -20°C

Big dye sequencing reaction
4.1- Equipment
 * PCR machine

4.2- Reagents

4.3- Other consumables

4.4- Procedure - enhancer: 			1 µl (if sequence GC rich) - big dye: 			1 µl - primer M13:		0.33 µl (10 µM stock=3.3 pmol) - 5X buffer:			1.0 µl - water:				4.67 µl - cleaned template:	2 µl
 * All the following steps must be made on ice
 * Prepare the following mix (quantities are for 1 tube):
 * put 8 µl of the mix in strip tubes
 * add 2 µl of template

lid temp: 110.0°C 96°C: 10 sec 50°C: 5 sec 60°C: 4 min 25°C: pause
 * Use a PCR machine for cycle sequecing (SWATI-SEQQ):

Cleaning of the sequencing reaction products by precipitation
5.1- Equipment
 * Centrifuge for 96 wells plates

5.2- Reagents
 * Na acetate 3M (store @ 4°C)
 * 85% ethanol (store @ 4°C)
 * 70% ethanol
 * formide dye (HIDI; AMRESCO 0606-100 ml)

5.3- Other consumables

5.4- Procedure - 1.9 µl of Na acetate 3M - 60 µl of 85% ethanol
 * Add to each 10 µl product:
 * Mix thoroughly (vortex ???)
 * keep at -20°C for 30 min
 * centrifuge for 45 min at 4000 rpm and 4°C (program 3; balance)
 * remove supernatant (invert tube on trash once)
 * add 150 µl of 70% ethanol
 * mix
 * centrifuge for 15 min at 4000 rpm and 4°C (program 4; balance)
 * remove all supernatant (invert tube on trash)
 * invert tube on paper tissue
 * centrifuge for 2 min at 500 rpm (program 7; no need to balance)
 * take out the tube and let it dry in the fume hood at room temperature for 10-15 min
 * put 20 µl of formide dye using multipipette (nasty chemical to manipulate in fume hood)
 * vortex thoroughly/spin/vortex/spin
 * transfer the 20 µl (multipipette) in a sequecing plate (careful on the order and remember that blocks of 4 are used)
 * put septum on top, press, tap once (do NOT mix)
 * keep on ice in aluminium rack
 * Heat 3 min at 95°C (SWATI/95-CST) to keep in single stranded form)
 * keep on ice in aluminium rack

Sequencing
6.1- Equipment
 * Applied Biosystems "3100-Avant Genetic Analyze"

6.2- Reagents

6.3- Other consumables
 * sequencing plate
 * septum

6.4- Procedure
 * launch "3100 Avant data coll"
 * ignore request for ZIP disk
 * insert sample names (BLOCKS of four)
 * Dye set: "z"
 * Mobility file: DT3100POP6(BDv3)v1.mob
 * Project name: 3100-Avant project1
 * Run module: XXXXLongRun
 * Analysis module: BC-3100APOP6SR_SeqOffFtOff.saz
 * Click OK
 * place the plate on a base and a cover on top, press down
 * press "Tray"
 * put plate, press evenly down, close door
 * select "JPG", click plate image (goes from yellow to green)
 * click on the green triangle pointing to the right
 * go to status or run view