Cloning Protocol

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Molecular cloning is similar to polymerase chain reaction (PCR) in that it permits the replication of DNA sequence. The fundamental difference between the two methods is that molecular cloning involves replication of the DNA in a living microorganism, while PCR replicates DNA in an in vitro solution, free of living cells.


Step 1: Phusion PCR


Do a 200 uL reaction for the insert (2 PCR tubes with 100 uL)

PCR Mix
Liquids Amounts
10 uM forward primer 10 uL
10 uM reverse primer 10 uL
2x phusion master mix (MM) 50 uL
10 ng total template 2 uL total
H2O up to 200 uL variable - top to 100 uL

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To calculate annealing temperature, use Tm, lower + 3⁰ C

PCR CYCLE
STEP TEMP TIME
Initial Denaturation 98⁰ C 30 s
25-35 cycles 98⁰ C 5-10 s
45-72⁰ C annealing 10-30 s
72⁰ C extension 15-30 s/kb
Final extension 72⁰ C 5-10 min
Hold 4⁰ C inf


Step 2: Digestion of vector


Digest 10 ug of vector to linearize while your PCR is ongoing. You can gel purify though if you're using 2 different restriction enzymes this is not required.

Try for 30 uL total.


5 ug vector
Amounts Liquids
1 uL Sph1
1 uL Xba1
3 uL Cutsmart
2 uL H20
22 uL DNA
30 uL TOTAL

Add

20 uL H2O

10 uL loading buffer (6x --> 1x)


Step 3: Purify PCR product


Combine 200 uL of PCR reaction in a 1.5 mL eppendorf tube.

Reagents used: salted EtOH--2% Potassium Acetate in EtOH (w/w)

To make: 0.237 g Potassium Acetate in 11.6 g (14.7 mL) of 100% EtOH

Gel Protocol


TAE 100 mL

Agarose 0.7 g

60 sec

shake

30 sec

add bromide

2 drops

shake

pour into mold


KI Gene PCR Product

200 uL DNA

20 uL Na Acetate pH 5.2 (1/10)

400 uL cold EtOH 100% (2x)


After warm room

Add 20 uL TE

Add loading buffer 4 uL of 6x

Run on Gel


Run all 20 uL in 2 separate wells on a 0.8-1.5% agarose gel (depends on the size of insert).

Take a photo of gel, and then cut bands using the higher wavelength setting on the UV lamp.

Purify large fragments (>200 bp) using regular QIAgen gel extraction kit. Purify small fragments (<100 bp) using QIAex II kit (has beads that bind DNA).

These are the vials with filter inserts

After DNA extract measure using nanodrop. Acceptable range is between 40-200 ng/uL


Step 4: Gibson Assembly Reaction


Recommended Amount of Fragments Used for Assembly
2-3 Fragment Assembly 4-6 Fragment Assembly Positive Control **
Total Amount of Fragments 0.02-0.5 pmols* 0.2-1 pmols* 10 uL
Gibson Assembly Master Mix (2X) 10 uL 10 uL 10 uL
Deionoized H2O 10-X uL 10-X uL 0
Total Volume 20 uL*** 20 uL*** 20 uL


Incubate samples at 50°C for 15 min when 2 or 3 fragments are being assembled, or 60 min when 4-6 fragments are being assembled.

To transform, dilute Gibson Assembly reaction 4-fold with H2O before transforming. Add 2 uL of diluted assembled product to competent cells. Proceed with transformation according to manufacturer's protocol.


GFP - Actin : 1866 bp · 650 g/bp·mol = 1212900 g/mol 114 ng/uL

mApple - Profilin : 1236 bp · 650 = 803400 g/mol 176 ng/uL

PSR5 : 9962 bp · 600 = 6475300 g/mol 55 ng/uL


0.7 % agar = 0.7 g

100 mL TEA

60 sec microwave

shake

30 sec microwave

add 2 drops bromide

pour in gel

takes ~ 45 minutes until ready


Step 5: Molecular Cloning


100 uL bacteria (competent E coli)

2 uL Mercampto EtOH

2 uL Gibson product after diluting to 4x


After transformation, Plate E coli on Agar

store upside down on agar plate in warm room for 24 hours

After 24 h prep falcon tube with 25 mL SB + 25 uL Carbinicilin

Prep 12 falcon-like tubes - 2 mL of the above solution goes into each tube, along with 1 bacterial colony. Use a pipette tip to pick up colony and leave entire tip in tube.

Label each tube 1:12 and transfer to large shaker table in warm room for 12+ hours, then transfer to 4°C


Step 6: MiniPrep