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

• Mix in DW:

- Tryptone: 5.0 g - yeast extract: 2.5 g - NaCl: 5.0 g

• 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:

- Ampicillin: dissolve 50 mg in about 100 µl DW - Xgal: dissolve 50 mg in about 250 µl DMSO

• 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

• 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:

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

• adjust pH to 7.0 w/ NaOH
• bring to volume:

for 1 l 500 ml 100 ml ddH2O(XXX) 980 ml 490 ml 98 ml

• autoclave on liquid cycle @ XXX°C for 20 min
• add autoclaved 1 M MgCl2

for 1 l 500 ml 100 ml 1 M MgCl2 10 ml 5 ml 1 ml

• add sterilized 2 M glucose

for 1 l 500 ml 100 ml 2 M glucose 10 ml 5 ml 1 ml

• aliquote in 2 ml tubes
• store at -20°C

1.7- DYT medium

• combine:

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

• 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

• Prepare a master-mix in a 0.5 ml tube containing (µl):

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)

• 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

• during this hour, finish preparing the LB plates (stored @ 37°C):

- prepare the appropriate number of Pasteur pipettes close tip and bend - clean bench using EtOH

• 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

• 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:

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

• 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

• 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):

lid temp: 110.0°C 37.0°C: pause 37°C: 30 min (incubate) 80°C: 15 min (denaturate) 25°C: pause

• 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

• All the following steps must be made on ice
• Prepare the following mix (quantities are for 1 tube):

- 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

• put 8 µl of the mix in strip tubes
• add 2 µl of template

• Use a PCR machine for cycle sequecing (SWATI-SEQQ):

lid temp: 110.0°C 96°C: 10 sec 50°C: 5 sec 60°C: 4 min 25°C: pause

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

• Add to each 10 µl product:

- 1.9 µl of Na acetate 3M - 60 µl of 85% ethanol

• 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