Cloning: Difference between revisions

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*aluminium rack for PCR tubes
*aluminium rack for PCR tubes
=== Media and plates ===
=== Media and plates ===
1.2- Reagents
'''Reagents'''
*Tryptone: Fisher BP1421-500
*[[LB]] (Luria-Bertami) agar plates
*yeast extract: Fisher BP1422-500
*[[IPTG]] stock solution (100 nM)
*NaCl: Sigma S-3014
*[[SOC]] medium
*MgCl2: ????
*[[DYT]] medium
*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
*Other consumables
#*gloves
**gloves
#*sterile (???) plates
**sterile (???) plates
#*pH paper
**pH paper
#*pasteur pipettes
**pasteur pipettes
#*DW
**DW
#[[LB]] (Luria-Bertami) agar plates
#[[IPTG]] stock solution (100 nM)
#[[SOC]] medium
#[[DYT]] medium


==Procedure==
==Procedure==
=== PCR products cloning ===
===Enriching the insert===
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.
The insert can come from another vector or loaded from a PCR product. PCR product should be clean by various [[Purification of DNA|purification methods]]  and there exists several protocols for [[PCR]]
 
===Restriction Digest===
Depending on the ends required for ligation, the ends of the insert and the vector may need to be prepared. For cloning methods like TA cloning, the Taq polymerase will add on the appropriate sticky ends. For other methods such as blunt-end ligation and sticky-end ligation, you will need digested vector and insert. See [[Restriction digest]].
 
===Ligation===
The process of ligating or joining ends of DNA together. This will take linear strand(s) of DNA and circularize the product to result in the desired plasmid. For more details see [[DNA ligation]].


2.1- Equipment
'''Reagents'''
*heating block
*[[T4 DNA ligase]]
*spinning centrifuge
*10x T4 DNA Ligase Buffer
*Purified, linearized vector (likely in H2O or EB)
*Purified, linearized insert (likely in H2O or EB)


2.2- Reagents
'''Method'''
*Vector: Find a good list via [[Vectors]]
#Mix reagents for a 10 μL reaction.
#Let reaction sit at Room Temperature for 30 minutes OR overnight at 16°C
#Denature ligase at 65°C for 10 minutes.
#Store at -20°C.
 
===Transformation===
Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. See [[Bacterial transformation]].
 
'''Reagents'''
*[[Competent cells]]: XL2-Blue (Stratagene 200150).
*[[Competent cells]]: XL2-Blue (Stratagene 200150).
**Learn how to make your own by [[Preparing_chemically_competent_cells]]
**Learn how to make your own by [[Preparing_chemically_competent_cells]]
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*ß-mercaptoethanol (ß-MAE)
*ß-mercaptoethanol (ß-MAE)


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


2.4- Ligation
===Screening===
See [[DNA Ligation]]
Either a [[Restriction_digest| check digest]] or [[Colony PCR]] will be a good way of screening your colonies. If the plasmid is of the correct size, submit for [[DNA sequencing]]
*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==
==Critical steps==
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{{#dpl:category=cloning|category=Protocol|titlematch=%:%}}
{{#dpl:category=cloning|category=Protocol|titlematch=%:%}}


===Specific Protocols===
 
*[[Knight:TOPO_TA_cloning]]
*[[General_Cloning_Protocol]]
*[[Duffy:LIC_Cloning]]
*[[Gateway_cloning_system]]


==Discussion==
==Discussion==

Revision as of 18:03, 12 January 2012

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

Reagents

  • LB (Luria-Bertami) agar plates
  • IPTG stock solution (100 nM)
  • SOC medium
  • DYT medium
  • Other consumables
    • gloves
    • sterile (???) plates
    • pH paper
    • pasteur pipettes
    • DW

Procedure

Enriching the insert

The insert can come from another vector or loaded from a PCR product. PCR product should be clean by various purification methods and there exists several protocols for PCR

Restriction Digest

Depending on the ends required for ligation, the ends of the insert and the vector may need to be prepared. For cloning methods like TA cloning, the Taq polymerase will add on the appropriate sticky ends. For other methods such as blunt-end ligation and sticky-end ligation, you will need digested vector and insert. See Restriction digest.

Ligation

The process of ligating or joining ends of DNA together. This will take linear strand(s) of DNA and circularize the product to result in the desired plasmid. For more details see DNA ligation.

Reagents

  • T4 DNA ligase
  • 10x T4 DNA Ligase Buffer
  • Purified, linearized vector (likely in H2O or EB)
  • Purified, linearized insert (likely in H2O or EB)

Method

  1. Mix reagents for a 10 μL reaction.
  2. Let reaction sit at Room Temperature for 30 minutes OR overnight at 16°C
  3. Denature ligase at 65°C for 10 minutes.
  4. Store at -20°C.

Transformation

Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. See Bacterial transformation.

Reagents

Method

Screening

Either a check digest or Colony PCR will be a good way of screening your colonies. If the plasmid is of the correct size, submit for DNA sequencing

Critical steps

Troubleshooting

Notes

See Also

Cloning Software

Protocols

General

Lab-specific protocols


Discussion

You can discuss this protocol.

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