Protocol

Abbreviation

Cloning_Toronto

Name

Cloning_Toronto

Laboratory name

University of Calgary

Type

cloning

Description

1. PCR Amplification
/Rxn
dH2O 34.1 µl
10x Pfx buffer 5.0 µl
50 mM MgSO4 1.0 µl 10 mM dNTP’s 1.5 µl
10x Enhancer 7.5 µl
genomic DNA (100ng/µl)0.4 µl
Pfx (2.5U/µl) 0.5 µl
Total Volume =50.0 µl
(PFx enzyme and buffer from Invitrogen)
*Make a master mix, aliquot 48 µl/rxn
and add: 1.0 µl forward primer (50 µM)
1.0 µl reverse primer (50 µM)
PCR Program:
1. 95°C 2:30 min.
2. 95°C 15 sec.
3. 53-56°C 30 sec.
4. 68°C 1 min./kb
5. Go To 2, Rep. 29x
6. 72°C 10 min
7. Hold 4°C


2. Run Large Gel – Verification of PCR product size
1% gel agarose gel in TAE
Large : 3 g /300 ml Small: 0.5 g/ 50 ml

1.Heat in microwave 4-5 min (~1 min for small gel).
2.For large gels let the solution cool for 20 min before pouring the gel, for small gels cast right away and it will take ~20 min to solidify
3.Load 5 µl of PCR product with 1.5-2 µl of 6x SYBR green loading dye
4.Run gel @ 220-240 ~30 min. (large gel), @ 120V ~20 min. (small gel)
5.View gel using gel doc (Settings: Program as per lab settings)


3. PCR Clean-up
For >12 PCR Products(QIAquick 96 PCR Purification Kit, handbook p. 20-22)

1.Transfer 45 µl PCR product to round bottom deep well (if the PCR reaction is 50 µl, there is not need to transfer into a new plate, PCR tubes/plate can be used).
2.Add 3:1 Buffer PM (135 µl).
3.Transfer to Qiagen Qiaquick 96 filter plate, pipette up/down a few times before transfer to mix sample and buffer.
4.Turn on vacuum for 5-7 min.
5.Add 900 µl Buffer PE, and vacuum for 5 min.
6.Repeat above.
7.Dispose of ethanol in waste tray, and vacuum 20-30 min.
8.Make sure all residual ethanol is gone by tapping on paper towel.
9.Elute with 50 µl EB buffer

Or For <12 PCR Products(QIAquick PCR Purification, handbook p. 19-20)
1.Transfer 45 µl PCR product to 1.5 ml microcentrifuge tube.
2.Add 5:1 Buffer PB (225 µl), mix by pipetting.
3.Transfer to QIAquick Spin Columns (pink) and centrifuge @ 14,000 rpm for 1min.
4.Add 750 µl Buffer PE, centrifuge @ 14,000 rpm for 1min.
5.Discard ethanol and centrifuge again @ 14,000 rpm for 1min.
6.Transfer column to a clean 1.5ml microcentrifuge tube.
7.Let sit at room temperature for ~30 min.
8.Elute with 50 µl of EB buffer.


4. Ligation Independent Cloning (LIC)Infusion Reaction – Annealing vector and insert
1.Dissolve BD-Infusion pellet with 8.5 µl of prepared vector(p15Tv-L, see below). Use 2 µl of the infusion/vector mix and add to each insert, therefore each infusion pellet can be used for 4 reactions (for a 96-well plate, use 22 pellets that will be pooled and centrifuged before dispensing in PCR tube/plate.
2.Add 2-4 µl of PCR target to the vector and mix by pipetting up/down at least 3-4 times
3.Incubate for 30 min at 28ºC, then place reactions on ice or freeze until use.


5. Transformation
1.Place PCR tube/plate containing ligation on ice ~10 min.
2.Add 80 µl of competent DH5α to each ligation.
3.Leave on ice for 10 min.
4.Heat shock @ 42°C for 45 sec.
5.Place on ice for 2 min.
6.Recover in 900 µl of SOC for 1 hr. @ 37°C with shaking.
7.Spin down @ 3000 rpm for 10 min.
8.Discard supernatant (flip plate only, do not tap, ~110 µl of media should remain at bottom). Use 55 µl to plate on LB + ampicillin (100 µg/ml) + sucrose (5%) plates. (be sure to incubate plates at 37°C to allow proper absorption).
9. Incubate plates 22 – 24 h @ 37°C (to have colonies big enough to do the screen).

6. Colony Screen

/Rxn
H2O 16.2 µl
Taq buffer (-MgCl2) 3.0 µl
25 mM MgCl2 2.4 µl
2 mM dNTP’s 3.0 µl
10x Enhancer 4.5 µl
T7 promoter (~600 µg/ml)0.3 µl
T7 terminator (~600 µg/ml)0.3 µl
Taq (5 U/µl) 0.3 µl
Total Volume= 30.0 µl



Make a master mix, aliquot 28 µl/rxn and add colony from transfomration to each well of the PCR plate with a tip. Pipet up/down to mix the colony to the master mix.
2 colonies per target are screened

PCR Program:
1.94°C 2 min.
2.94°C 30 sec.
3.50°C 30 sec.
4.68°C 1 min./kb
5.Go To 2, Rep. 29x
6.72°C 10 min
7.Hold 4°C


7. Run Large Gel – Verification of positive clones
As previously reported (see aabove).


8. Grow
1.Pick positive colony for each clone, and add to 1 ml of LB + amp (100 µg/ml) in deep well plate.
2.Shake O/N @ 37°C.

9. Plasmid Prep of Positive Clones (QIAprep 96 Turbo Miniprep Kit, handbook p. 33-35)
1.Spin O/N grown plate for 10 min. @ 3000 rpm.
2.Discard supernatant.
3.Add 250 µl P1 buffer and vortex to resuspend pellet.
4.Add 250 µl P2 buffer (lysis buffer) and shake slowly, let sit for no more than 5 min.
5.Add 350 µl N3 buffer (neutralization buffer) and shake slowly.
6.Transfer (850 µl) to Qiagen Turbofilter plate (white), with Qiaprep plate (blue) beneath.
7.Vacuum for 3-5 min.
8.Discard TurboFilter, and place Qiaprep plate on top.
9.Vacuum for ~10 min.
10.Add 900 µl PE buffer, and vacuum 3 min.
11.Repeat above step.
12.Let sit at room temperature for ~30 min.
13.Elute into a shallow round bottom plate with 100 µl buffer EB.
14.Label plate and store at -20°C or aliquot plasmids

Or
For individual minipreps (Qiagen Manuel, pg. 22- 23)
1.Grow a colony of the positive clone in 2ml of LB+amp (100 µg/ml) in a 14 ml falcon tube overnight at 37°C.
2.The next day, centrifuge the tubes at 3000 rpm for 10 min.
3.Discard supernatant.
4.Add 250 µl P1 buffer and vortex to resuspend pellet.
5.Tranfer the sample to a 1.5ml microcentifuge tube
6.Add 250 µl P2 buffer (lysis buffer) and shake slowly,let sit for no more than 5 min.
7.Add 350 µl N3 buffer (neutralization buffer) and invert gently
8.Centrifuge for 10 min @ 13,000rpm
9.Apply the supernatants form step 8 to the QIAprep spin column by decanting or pipetting
10.Centrifuge for 1min @ 13,000rpm
11.Wash the column with 500 µl of Buffer PB and centrifuge for 1 min.
12.Wash the column with 750 µl Buffer PE and centrifuge for 1 min.
13.Discard the flow-through and centrifuge for an additional 1 min to remove the residual buffer.
14.Place the column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl Buffer EB and centrifuge for 1 min.
15.Label tube(s) and store at -20°C


Preparation of p15TV-L vector please refer to this website for vector information
http://www.thesgc.org/SGC-WebPages/StructureDescription/M&M/Vectors/p15TV-L.pdf

1.Transform p15TV-L in E. coli DH5α competent cells.
2.Grow 3 x 4 ml overnight cultures of p15Tv-L from single colony in LB + amp (100 µg/ml) in 14 ml Falcon tubes.
3.Miniprep using Qiagen spin columns (QIAprep Spin Miniprep Kit, handbook p. 22-23), using 4 ml of culture per column.
4.Elute with 50 µl of Buffer EB per column.
5. Digestion:

p15Tv-L 120 µl
10x NEB #2or4 60 µl
BseRI 15 µl
H2O(RNAse/DNAse- free) 405 µl
incubate 1.5 h @ 37C

Add
10x NEB #2or 4 10 µl
BseRI 5 µl
H2O (RNAse/DNAse- free) 85 µl
incubate 1.5 h @ 37C
Total Volume =700 µl

6.Transfer to 15 ml conical falcon tube, add 3.5 ml of PB buffer
7.Purify by transferring 700 µl each to 6 Qiagen spin columns (QIAquick PCR Purification Kit, handbook p. 19-20).
8.Elute with 50 µl EB per column. Pool the samples and keep at -20oC until use.
9.Visualize digestion on 1% agarose gel (two bands should appear at ~8Kb and 2Kb).