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)
|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|
To calculate annealing temperature, use Tm, lower + 3⁰ C
|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|
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.
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
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
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