Cloning

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Contents

General Procedure

This should be a consensus protocol. See the bottom of this article for specific protocols.

Materials

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. Other consumables
    • gloves
    • sterile (???) plates
    • pH paper
    • pasteur pipettes
    • DW
  2. LB (Luria-Bertami) agar plates

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:
Reagent for 1 L 500 mL 100 mL
tryptone 20 g 10 g 2g
yeast 5g 2.5 g 0.5 g
NaCl 0.5 g 0.25 g 0.05 g
250 nM KCl 10 mL 5mL 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

Procedure

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

2.3- Other consumables

  • 0.3 µl sterile ??? tubes
  • DW

2.4- Ligation See DNA 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

Best protocols would be under Purification of DNA.

General Procedure goes as follows

  1. Add 1/10 volume of 3M sodium acetate and 2-3 volumes of 100% Ethanol
  2. Mix and freeze overnight in -20.
  3. Spin at full speed in a standard microcentrifuge at 4 degrees for 30 minutes.
  4. Dry the pellet.
  5. Add your desired quantity of water. Vortex and spin down to resuspend.

Sequencing

See Sequencing DNA

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

Critical steps

Troubleshooting

Notes

See Also

Specific Protocols

Discussion

You can discuss this protocol.

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