Cloning: Difference between revisions

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==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]]


2.1- Equipment
===Ligation===
*heating block
Just see [[DNA ligation]]
*spinning centrifuge


2.2- Reagents
====Reagents====
*Vector: Find a good list via [[Vectors]]
*[[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====
#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
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
===Screening===
*launch "3100 Avant data coll"
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]]
*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==

Revision as of 20:52, 29 December 2011

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

  • 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

Ligation

Just 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

Specific Protocols

Discussion

You can discuss this protocol.

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