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HOW TO ... Clone with the promega PGEM-T kit
{{back to protocols}}
==General Procedure==
This should be a [[Help:Consensus protocol|consensus protocol]]. See the bottom of this article for specific protocols.  


Rutgers, November 2004
==Materials==
 
===Equipment===
== Media and plates ==
1.1- Equipment
*gas burner
*gas burner
*rotating plate
*rotating plate
*aluminium rack for PCR tubes
*aluminium rack for PCR tubes
=== Media and plates ===
'''Reagents'''
*[[LB]] (Luria-Bertami) agar plates
*[[IPTG]] stock solution (100 nM)
*[[SOC]] medium
*[[DYT]] medium


1.2- Reagents
*Other consumables
*Tryptone: Fisher BP1421-500
**gloves
*yeast extract: Fisher BP1422-500
**sterile (???) plates
*NaCl: Sigma S-3014
**pH paper
*MgCl2: ????
**pasteur pipettes
*glucose: ???
**DW
*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
==Procedure==
*gloves
===Enriching the insert===
*sterile (???) plates
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]]
*pH paper
*pasteur pipettes
*DW


1.4- LB (Luria-Bertami) agar plates
===Restriction Digest===
*Mix in DW:
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]].
- 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)
===Ligation===
*Dissolve 238 mg IPTG (sigma I-6758) in 10 ml DW
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]].
*store in 1 ml aliquotes at -20°C


1.6- SOC medium
'''Reagents'''
*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)
*[[T4 DNA ligase]]
*2M glucose stock: dissolve 18 g glucose into 50 ml (final volume) ddH2O (xxx) and filter-sterilize into sterile 50 ml tube
*10x T4 DNA Ligase Buffer
*250 mM KCl stock: dissolve 1.86 KCl in 100 ml ddH2O (XXX)
*Purified, linearized vector (likely in H2O or EB)
*combine:
*Purified, linearized insert (likely in H2O or EB)
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
'''Method'''
*combine:
#Mix reagents for a 10 μL reaction.
500 ml 100 ml
#Let reaction sit at Room Temperature for 30 minutes OR overnight at 16°C
tryptone 8.0 g 1.6 g
#Denature ligase at 65°C for 10 minutes.
yeast 5.0 g 1.0 g
#Store at -20°C.
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 ==
===Transformation===
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.
Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. See [[Bacterial transformation]].


2.1- Equipment
'''Reagents'''
*heating block
*[[Competent cells]]: XL2-Blue (Stratagene 200150).
*spinning centrifuge
**Learn how to make your own by [[Preparing_chemically_competent_cells]]
 
*[[SOC]] medium
2.2- Reagents
*Vector: Promega pGEM-T cloning kit
*Competent cells: XL2-Blue (Stratagene 200150)
*SOC medium
*ß-mercaptoethanol (ß-MAE)
*ß-mercaptoethanol (ß-MAE)


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


2.4- Ligation
===Screening===
*Prepare a master-mix in a 0.5 ml tube containing (µl):
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]]
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)
==Critical steps==
*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):
==Troubleshooting==
- prepare the appropriate number of Pasteur pipettes close tip and bend
==Notes==
- clean bench using EtOH
==See Also==
*when the incubation is complete, spread 145 µl of SOC-CC onto plate
===Cloning Software===
*let soak 10 min @ 37°C
{{#dpl:category=cloning|category=software}}
*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
* [http://openwetware.org/wiki/Genome_compiler Genome Compiler]: watch these short [http://www.genomecompiler.com/cloning-easier-than-ever/ tutorial videos] to learn how to use the Cloning Wizards (simulate digest and ligate with restriction enzymes or by PCR).
*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 ==
==Protocols==
3.1- Equipment
===General===
*material for gel electrophoresis
{{#dpl:category=cloning|category=Protocol|nottitlematch=%:%}}
*PCR machine


3.2- Reagents
===Lab-specific protocols===
*Shrimp alkaline phosphatase (SAP), stock solution 1 U/µl [removes P from 3' end of DNA]
{{#dpl:category=cloning|category=Protocol|titlematch=%:%}}
*exonuclease I, stock solution 10 U/µl [removes single strand DNA]


3.3- Other consumables
===Online Courses===


3.4- Cleaning
[http://www.slideshare.net/Rutial/restriction-ligation-course Restriction Ligation cloning method] - a practical guide for this cloning technique.  
*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
==Discussion==
 
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
 
==Protocols==
===Lab-specific protocols===
{{#dpl:category=Cloning|category=Protocol|titlematch=%:%}}


{{stub}}
You can [[Talk:{{PAGENAME}}|discuss this protocol]].
[[Category:Protocol]] [[Category:Cloning]]
[[Category:Protocol]]
[[Category:Cloning]]
[[Category:DNA]]
[[Category:In vitro]]

Latest revision as of 03:34, 21 January 2016

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

  • Genome Compiler: watch these short tutorial videos to learn how to use the Cloning Wizards (simulate digest and ligate with restriction enzymes or by PCR).

Protocols

General

Lab-specific protocols

Online Courses

Restriction Ligation cloning method - a practical guide for this cloning technique.


Discussion

You can discuss this protocol.

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