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
