Cloning
back to protocols | ||
General Procedure
This should be a consensus protocol. See the bottom of this article for specific protocols.
Materials
Equipment
- gas burner
- rotating plate
- aluminium rack for PCR tubes
Media and plates
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
- Other consumables
- gloves
- sterile (???) plates
- pH paper
- pasteur pipettes
- DW
- LB (Luria-Bertami) agar plates
- IPTG stock solution (100 nM)
- 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
- DYT medium
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
- Vector: Find a good list via Vectors
- Competent cells: XL2-Blue (Stratagene 200150).
- Learn how to make your own by Preparing_chemically_competent_cells
- SOC medium
- ß-mercaptoethanol (ß-MAE)
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- Bacterial 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
- Add 1/10 volume of 3M sodium acetate and 2-3 volumes of 100% Ethanol
- Mix and freeze overnight in -20.
- Spin at full speed in a standard microcentrifuge at 4 degrees for 30 minutes.
- Dry the pellet.
- 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
- Knight:TOPO_TA_cloning
- General_Cloning_Protocol
- Duffy:LIC_Cloning
- Gateway_cloning_system
- Cloning_Checklist
- Cloning_and_sequencing
- Cloning_Protocol
- Knight:TOPO TA cloning
- Wikiomics:Cloning in silico
- Wikiomics:Site Directed Mutagenesis
- Duffy:LIC Cloning
- Rutgers:DNA Ligation
- Rutgers:Transformation
- Milo:No background cloning protocol
Discussion
You can discuss this protocol.