Protocol
1 DNA double digestion protocol
Materials:
DNA sample(s) in water or TE buffer
10x digestion buffer
Restriction enzyme s (EcoRI or SpeI or XbaI or PstI)
DNA loading buffer (if electrophoresis is subsequent)
Agarose gel 1.5% (or different depending on expected band sizes)
Procedure:
1. Test the concentration of the DNA sample(s).
2. Pipet the following into a microfuge tube:
20uL reaction system 50uL reaction system
DNA around 1ug around 2.5ug
10x Digestion buffer 2uL 5uL
1st Enzyme 1-1.5uL 2.5-4uL
2nd Enzyme 1-1.5uL 2.5-4uL
ddWater Rest of volume Rest of volume
3. Incubate at recommended temperature (37.0 degrees) for 2 or 4 hours
(0.5~2hfor enzymes of NEB, 4h for enzymes of Takara).
4. Take 2 to 5 uLof the digested sample, add loading buffer, and run it on the
agarose gel to check the result, or take the entire sample to run to extract a
wanted fragment).
Tips:
1. DNA:
For identification of DNA, use 0.4 ug/uL DNA; (or 2uL from a nice DNA
mini prep)
For cloning, 1ug/uL DNA is enough.
2. Buffer: we’ d better use the buffer that comes with the enzyme, which
means buffers from other company may cause some abnormal results.
3. Enzyme: the maximum volume that an enzyme can be used is 1/10 of the
total reaction volume (example: 2 uL for 20 uL reaction system). If you want
to do overnight digestion, add less enzyme (example: 1 uL for 20 uL reaction
system). It is necessary to point that too many enzymes will reduce the
efficiency of enzyme digestion with glycerol in it.
4. Gel: make sure to run the uncut DNA as a control along with the digested
DNA sample(s). And, always run a DNA marker!
References:
*Current protocols in molecular biology (3.1.1-3.1.2)
2 DNA Gel extraction
Here is a suggested protocol; this protocol can be used to purify a wide
range of DNA fragments with recoveries of >80%. The bolded should be
noticed for a nice DNA extraction.
1. Excise gel slice containing the DNA fragment using a clean scalpel or razor
blade.
Cut as close to the DNA as possible to minimize the gel volume. Place the gel
slice into a pre-weighed 1.5 ml tube and weigh. Record the weight of the gel
slice.
2. Put EB (elution buffer) or ddwather at 65 degree water bathing.
3. Add a 3:1 volume of Solution Buffer to the gel slice (volume:weight) (e.g.,
add 300 ul of Binding Buffer for every 100 mg of agarose gel). Incubate the
gel mixture at 65 degree for 5 min at least until the gel slice is completely
dissolved.
Mix the tube by inversion every few minutes to facilitate the melting process.
Check the color of the solution. Ayellow color indica tes an optima l pH for
DNAbinding. If the color of the solution is orange or violet, add 10 ul of 3 M
sodium acetate, pH 5.2 solution and mix. The color of the mix will become
yellow.
4. Pour the solution to a fresh adsorption column. Centrifuge at 13000rpm
for 1 min.Pour off the liquid in the collection tube. For critical samples,
repeat the operation above for 2 or 3 times.
5. Add 650 ul washing buffer (WB) before centrifugation at 13000 rpm for 1
min.Pour off the liquid into beaker.
6. Centrifuge at 13000rpm for 10 min to spin the ethanol down.
7. Put the column into a fresh EP tube. If necessary air-dry the pellet for
10-15 min to avoid the presence residual ethanol in the purified DNA solution.
Residual of ethanol in the DNA sample may inhibit downstream enzymatic
reactions.
8. Add 30-50 ul elution buffer (EB) to elute the DNA.
9. Get 5 ul of the eluted sample to identify with electrophoresis.
Notes:
1. Extract the gel as soon as you excise the gel slice.
2. If the purified DNA will be used for cloning, avoid UV damage of the DNA
by minimizing the UV exposure to a few seconds or keeping the gel slice on a
glass or plastic plate during UV illumination.
3. If a large amount of DNA is purified or if the volume of the binding reaction
is greater than 1.5 ml increase the incubation time of the binding step to 15
min.
References:
*Current protocols in molecular biology
3 Recombinant plasmid construction
|
experimental material |
|
|
template gene |
1ul |
|
sense Primer |
0.5ul |
|
antisense primer |
0.5ul |
|
dNTP |
1ul |
|
enzyme |
1ul |
|
2*buffer solution |
25ul |
|
Water |
21ul |
1)primer design
2) Obtain the target gene fragment
a. Run PCR
Put the mixture into the PCR instrument, run related program
3) Obtain the enzyme cutting vector
a. Enzyme digestion
I. Raw materials: Sall enzyme, NotI enzyme , water, buffer, plasmid
II. inject enzyme, buffer and water into the tube containing plasmids.
III. The tube was put into a 37 ° C water bath and digested for 4 hours.
IV. Remove the tube from the water bath, recycle the gel and put it at 4℃.
b. Agar Gel electrophoresis
c. Gel recycling
4) Ligation
Meterial: recombinase, the enzyme cutting vector, the target gene fragment and H2O
I. Add DNA fragments and cleaved plasmid as 4:1 into test tube.
II. Add recombinase and water. Place it into the 37 ° C water bath for 30 min.
5) Transformation (shake bacteria, coated plate, pick bacteria, elution)
a. Making a medium
I. Mix 1 gram of Tryptone, 1 gram of NaCl, 0.5 g of yeast and add water to 100 ml.
II. Mix the solution with a magnetic stirrer.
III. Distribute the stirred solution into different test tubes and sterilize them with autoclave for 30 min
IV. Remove the solution, store some under 4 ° C and add others with agarose powder and store at 4 ° C after it solidify
b. Transformation
I. Take the medium from 4 ° C
II. Add the plasmid into the competent cell. Ice for 30 min, heat for 45 seconds and - Ice again for 5 min or more
III. Place the test tube on a shaker at 37 ° C and 150 rpm for 1 hour
IV. Take the competent bacteria from the shaker
V. Swab the competent bacteria on the medium
VI. Immerse sticks into alcohol twice
VII. After the stick cooled down, use it to spread the bacteria by drawing ‘z’ shape on the medium.
VIII. After that, seal the medium, mark the corresponding date and bacteria, culture the medium in the incubator overnight at 37 ° C (up to 12 hours).
C. Pick the bacteria
I. Remove the cultured dish from incubator
II. Use the tip of the gun to spot the bacteria to remove the bacteria
III. Throw the tip that has the bacteria into liquid medium and put it into the shake at 150 rpm overnight.
IV. Extract the cultured bacteria from the cultured medium
d. obtain the plasmid
I. Add 500 μl of equilibration solution BL to the adsorption column CP3, centrifuge for 1 minute at 12000 rpm, discard the waste from the collection tube, and return the adsorption column to the collection tube.
II. Take 1-5 ml of bacterial solution, add to the centrifuge tube, centrifuge 1 minute at 12000 rpm, try to remove the supernatant.
III. Add 250 μl of solution P1 to the centrifuge tube where the bacteria are left to precipitate, use the gun to stir until no precipitate can be observed by naked eye
IV. Add 250 μl of P2 to the centrifuge tube, gently flip the tube 6-8 times. So that the cell lysis.
V. Add 350 μl to the centrifuge tube P3, immediately gently flip the tube up and down 6-8 times until fully mixed, this time there will be white flocculent precipitation.
VI. Transfer the supernatant collected from the previous step to the suction column P3 and place it in the collection tube (do not draw the precipitate).
VII. Use 12000rpm to centrifuge 30-60 seconds, drained the waste in the collection tube, place the adsorption column into the collection tube.
VIII. Add 600 μl of rinse PW solution to the adsorption column, centrifuge at 12,000 rpm for 30-60 seconds, discard the waste in the collection tube, and place the column into the collection tube.
IX. Repeat step 7
X. Centrifuge adsorption column CP3 again at 12,000 rpm for 2 minutes, drained the waste.
XI. Place the adsorption column in a clean centrifuge tube, add 50-100 μl of the elution buffer to the middle of the adsorbent membrane, allow the mixture to stand for 2 minutes at room temperature, centrifuge at 12000 rpm for 2 minutes and collect the plasmid solution into the centrifuge tube.
e. Ligation
Material: recombinase, the enzyme cutting vector, the target gene fragment and H2O
I. Add DNA fragments and cleaved plasmid as 4:1 into test tube.
II. Add recombinase and water. Place it into the 37 ° C water bath for 30 min.
f. Sequencing
II. Send the plasmid for sequencing after observing the line.
4. Transformation protocol
Materials:
Plasmid samples or ligation product;
Commercially competent cells;
LB non-antibiotic liquid meium;
LB antibiotic agar plates
Procedure:
1. Get the competent cells from -70 degree, and wait for its fusion. 30-50 μ
l of competent E.coli cells for each sample. Put microcentrifuge tubes to
chill on ice for at least 2 min.
2. Add 2 - 3 ul of each plasmid sample or all the ligation product into the competent cells in the microcentrifuge tubes. Mix and incubate on ice for 30
min.
3. Heat pulse for 90 sec, at 42 degree. Put back to ice and incubate for 5 min.
4. Add 200 uL LB non-antibiotic liquid medium into each microcentrifuge
tube. Shake the microcentrifuge tubes in shaker, at 37 degree, for 30 min to
recover.
5. Plate 150 uL of the liquid medium with transformed cells immediately, on
prewarmed LB antibiotic agar plates. Incubate overnight at 37°C for 10-14h.
Tips:
-All procedures are performed on ice.
- Make sure the cells are not left at ambient temperature for more than 5min as thiswill significantly decrease the transformation efficiency.
- When got out from the shaker, the competent cells ma y form pellet in
the microcentrifuge tubes. You need to resuspend the cells before plating.
References:
*Current protocols in molecular biology.