Protocols
1-Transformation of E.coli (DH5a, TOP10, BL21) with plasmid DNA
1. Thaw competent cells into ice
2. Add 50-100ng of plasmid DNA into the cell culture
3. Put it in ice for 15-30 minutes
4. Heat shock at 42℃ for 45 – 90 seconds
5. Put the cells into ice for 2 minutes
6. Add 1ml SOB
7. Incubate at 37℃ for 60 – 90minutes with shaking at 250rpm
8. Spead 100µl culture onto an LB agar plate with antibiotics
9. Incubate the plate overnight at 37℃
2-Restriction Enzyme Cutting (Double digestion)
Reaction mixture with total volume of 20µl
1µl DNA
1µl enzyme 1
1µl enzyme 2
0.2µl BSA
2µl NEB buffer
14.8µl ddH2O
Buffer chart for enzyme 1 and 2
http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp#.UFAsQI0ge80
Protocol
Incubate the mixture at 37℃ for 30 minutes
3- Gel electrophoresis
Set gel
1. Dissolve x g of agarose powder in to 100x ml of 1X TAE buffer ( x depends on the size of the gel)
2. Microwave for 1 minute
3. Add 5x µl of RedSafeTM into the solution
4. Pour the solution to the gel rack with a comb inserted. Let the gel to solidify by cooling under room temperature
Run gel
1. Transfer the gel into a gel tank once it is solidified with the wells pointing to the negative electrode
2. Check whether the TAE buffer covers the gel for about 1mm
3. Add the DNA with loading dye mixed into the wells
4. Add 2µl of KAPA DNA ladder into a well
5. Connect the tank with the electrodes and set the power supply to 150V.
6. Let the gel to run for 40 minutes.
4- Miniprep (using iNtRON DNA-spin Plasmid DNA Purification Kit with modifications)
1. Pick a single colony from a freshly streaked bacterial plate and use it to inoculate LB plus an appropriate antiboitic. Incubate the culture overnight with shaking.
2. Harvest 3-5ml of bacterial culture by centrifugation at 13,000rpm for 30 sec at RT and discard the supernatant.
3. Resuspend the pellet in 250µl of Resuspension buffer, vortexing until no clumps of the cell pellet remain.
4. Add 250µl of Lysis buffer to resuspended cells. Close tube and gently mix by inverting the tube several times. Do not vortex and do not exceed 5 min for lysis time.
5. Add 350µl of Neutralization buffer and gently mix by inverting the tube several times.
Note : Do not vortex. The solution should become cloudy and a flocculent precipitate should form. To improve the yield, place the tube at 4℃ for 5minor use chilled Neutralization buffer to enhance the precipitation.
6. Centrifuge at 13,000 rpm for 10 min at 4℃. While waiting for the centrifugation, insert a column into collection tube.
7. After centrifugation, transfer supernatant promptly into the column.
8. Centrifuge at 13,000 rpm for 60 sec. Remove the column from collectiontube, discard filtrate in collection tube. And then place the spin column back in the same collection tube.
9. Add 700µl of Washing buffer B, centrifuge at 13,000 rpm for 60 sec.Discard filtrate in the collection tube and place the spin column back in thesame collection tube. Repeat using 500µl of Washing buffer B.
10. Centrifuge at 13,000 rpm for 60 sec to dry the filter membrane.
11. Put the column into a clean and sterile centrifuge tube. Add 30µl of Elutionbuffer to the upper reservoir of the column, and let it stand for 2min. Then, centrifuge the tube assembly at 13,000 rpm for 60sec.
5- Ligation
Reaction mixture with a total volume of 20μl
2 μl 10X ligation buffer
1 μl T4 DNA ligase
17 μl of insert , vector and ddH2O
Allow the reaction to be taken place at 16oC overnight, or room temperature for 2 hours.
6- PCR (Using Phusion)
Pipetting instructions (in order):
50 µl reaction
add to 50 µl ddH2O
10 µl 5x Phusion HF/GC Buffer
1 µl 10 mM dNTPs
0.5 µl primer A
0.5 µl primer B
1 µl template DNA
0.5 µlPhusion DNA Polymerase
Protocol
1. Set up the reaction mixture while adding the DNA polymerase at the second last and template DNA at the last.
2. Place them into a thermo cycler and run the cycle program file
7- PCR purification (Using PCRquick-spin PCR Product Purification Kit with modifications)
1. Add 500µlof Binding buffer to PCR product (up to 50µl) in 1.5ml microcentrifuge tube and mix well.
2. After mixing, incubate at room temperature for 1min.
3. During the incubation time, place a spin column in a provided 2ml collection tube.
4. Load the sample to the spin column and centrifuge at 13,000rpm for 1min. Discard the flow-through after centrifuging and place the spin column back in the same 2ml collection tube.
5. Add 700µl of Washing buffer to column and centrifuge at 13,000rpm for 1min. Discard the flow-through after centrifuging and place the spin columnback in the same 2ml collection tube. Repeat with using 500µl of Washing buffer.
6. Centrifuge for 1 min at 13,000rpm to dry the spin membrane.
7. Put the column into a clean and sterile centrifuge tube. Add 30µl of Washing buffer of Elution buffer to the upper reservoir of the column, and let it stand for 2 mins. Then, centrifuge the tube assembly at 13,000 rpm for 60sec
8-Gel purification (Using GenScript QuickClean II Gel Extration Kit with modifications)
1. Excise the DNA band from the agarose gel with a clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose.
2. Weigh the gel slice. Then Add 3 volumes of Binding Buffer II to 1 volume of gel slice (100mg≈100ul),the gel slice should not be more than 400mg per test.
3. Incubate at 55℃ for 10 minutes with occasional vortexing or until the gel slice has been completely.
5. Transfer the sample to Spin column, centrifuge at 6,000×g for 1 min. Discard the flow through until the sample is processed completely. If the sample volume is more than 750 µl, load and centrifuge again using the same column.
6. Add 500 µl Binding Buffer II to Spin column, centrifuge at 12,000 ×g for 60s. Discard all flow through liquid.
7. Add 750 µl Wash Buffer to Spin column, centrifuge at 12,000 ×g for 60s. Discard all flow through liquid.
8. Centrifuge at 12,000 ×g for an additional 1 minute and transfer the Spin column to a sterile 1.5ml microcentrifuge tube.
9. Add 30 µl Elution Buffer to the Spin column and let it stand for 2 minutes at room temperature.
10. Centrifuge at 12,000 ×g for 1 minute. The buffer in the micro-centrifuge tube contains the DNA.
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