Experiment
1
Experiment
purpose: Insert the NAMPT gene into a mammalian expression vector (pCDH) to express
the NAMPT protein
Method: Molecular
cloning
STEP
1: Get the CDS sequence of the NAMPT gene from NCBI
STEP 2: Use SnapGene to construct
the new plasmid
STEP 3: PCR reaction
|
Reagent |
Volume ( µL) |
|
PCR grade water |
21 |
|
KOD One™ PCR Master Mix |
25 |
|
10 µM Primers Forward/Reverse |
1.5 |
|
Template |
1 |
|
Total volume |
50 |
PCR cycle
1) Denaturation: Break the hydrogen bonds between DNA bases
to melt the DNA double strands to produce single-stranded DNA.
2) Annealing: Lower the temperature low enough to allow
the oligonucleotide primer to bind to the DNA template.
3) Extension: During extension, DNA polymerase will
synthesize new double-stranded DNA.
|
Process |
Temperature(℃) |
Time(s) |
Cycle times |
|
Pre-denaturation |
98 |
180 |
1 |
|
denaturation |
98 |
10 |
30 |
|
Annealing |
65 |
5 |
|
|
Elongation |
68 |
20 |
|
|
Elongation 2 |
65 |
300 |
1 |
|
preservation |
4 |
- |
1 |
STEP 4: Empty vector double
digestion for linearization
Reaction mix: 20ul system
Ingredients:
|
Reagent |
Volume ( µL) |
|
pCDH vector |
3 |
|
BamHl |
0.5 |
|
EcoRI |
0.5 |
|
ddH2O |
16 |
|
Total volume |
20 |
Condition: PCR tube, 37℃, overnight
STEP 5: Carrier double digestion linearization
1. Weigh 0.3g agarose powder and add it to 30 mL TAE
solution. Microwave oven to melt the agarose powder until the solution is clear and transparent.
2. Pour the agarose gel solution into the gluing tank and
insert the comb.
3. After solidification for about 30 minutes, take out
the nucleic acid glue and use or store it in TAE solution immediately for later
use.
STEP 6: Agarose Gel Electrophoresis
Observe nucleic acid gel electrophoresis.
STEP 7: Gel extraction
1.
Cut gel to 1.5ml EP tube.
2.
Add NT1 buffer (200 µl/mg).
3.
Dissolve the gel in the buffer in a metal bath at 50℃ for 5-10min
4.
Load the sample into the column.
5.
Use a centrifuge (11000xg,30s) to centrifuge the DNA from the buffer.
6.
Change a tube for a column, add NT3 buffer (700 µl/mg).
7.
Use centrifugal machine (11000xg,30s) to centrifuge the DNA from the buffer.
8. Repeat steps 6 and 7.
9.
Dry silica membrane (11000xg, 1min).
10. Change tube and add 50 µl ddH₂O, then elute the
DNA into the ddH₂O by Centrifuge (11000xg,30s).
STEP 8: Concentration determination
1.
Add 2 µl to the NanoDrop 2000.
2.
Determinate concentration.
STEP 9: The carrier is connected
to the fragment
1. Mix Enzyme mix (5 µl), Cut vector (1 µl), NAMPT fragment
(3 µl), ddH2O(1 µl)
2. Incubate at 50℃ for
15 min
(Immediate use it for transformation; If subsequent
operations cannot be carried out immediately, the reaction sample should be
stored at -20℃)
STEP 10: Transformation
1.
Operation Method
1) Take 50 µL of competent cells
melted in an ice bath, add the target DNA, mix them gently, and place in an ice
bath for 30 minutes.
2) Heat in a 42℃ water bath for 45 seconds, then quickly
transfer the tube to an ice bath for 2 minutes without
shaking the centrifuge tube.
3) According to the experimental requirements
(transformation of plasmids and recombinant ligation products), pipette different
volumes of transformed competent cells and add them to LB agar medium
containing corresponding antibiotics, and spread the cells evenly. Place the
plate at 37℃ until the liquid is absorbed, invert
the plate, and culture overnight at 37℃.
2.
Precautions
1) Freshly thawed cells have the highest transformation
efficiency.
2)
Avoid repeated thawing.
3)
Avoid pipetting.
4) The whole operation should be
gentle.
STEP 11: Colony PCR-monoclonal identification
1. Picked a single colony into the medium containing
100ul ampicillin resistance, cultured on a shaker at 37℃ for 1 hour.
2. Same as step 3, perform a PCR (10ul system).
3. Same as step 5+6, Agarose
Gel Electrophoresis.
4. Shake positive
colonies tube 7ml culture medium overnight culture.
5.
Plasmid extraction.
STEP 12: Plasmid extraction
1. Column equilibration step: Add 500℃L of
equilibrium liquid BL to the adsorption column CP3 (the adsorption column is
placed in the collection tube), centrifuge at 12,000 pm (~13,400xg) for 1 min,
pour off the waste liquid in the collection tube, and remove the adsorption.
Return the column to the collection tube.
2. Take 1-5 mL of overnight cultured bacterial solution,
add it to a centrifuge tube, use a conventional desktop centrifuge, centrifuge
at 12,000 rpm (~13,400Xg) for 1 min, and suck up
the supernatant as much as possible (when there is a lot of bacterial
solution, you can centrifuge the bacteria several times).
3. Add 250µL of solution P1
(please check whether RNase A has been added) to the centrifuge tube with the
bacterial pellet left, and use a liquid dispenser or a vortex shaker to suspend
the bacterial pellet thoroughly. (If there are bacteria blocks that are not
thoroughly mixed, the lysis will be affected, resulting in low extraction
volume and purity).
4. Add 250µL solution P2 to the
centrifuge tube, and gently turn it up and down 6-8
times to fully lyse the bacteria (Mix gently, do not shake vigorously,
so as not to break the genomic DNA, resulting in genomic DNA fragments mixed in
the extracted plasmid. At this time, the bacterial solution should become clear
and viscous, and the time used should not exceed 5 minutes to avoid damage to
the plasmid. If it does not become clear, it may be due to too much bacteria
and incomplete lysis, and the amounts of bacteria should be reduced).
5. Add 350µL of solution P3 to
the centrifuge tube, turn it up and down gently 6-8 times immediately, and mix
well. At this time, a white flocculent precipitate
will appear. Centrifuge at 12,000 rpm (~13,400Xg) for 10 min. (P3 should be
mixed immediately after adding to avoid local precipitation. If there are tiny
white precipitates in the supernatant, centrifuge again and take the
supernatant).
6. Use a pipette to transfer the supernatant collected in
the previous step to the adsorption column CP3 (the adsorption column is placed
in the collection tube), and be careful not to suck
out the precipitate as much as possible. Centrifuge at 12,000 rpm
(~13,400Xg) for 30-60 sec, discard the waste liquid in the collection tube and
put the adsorption column CP3 into the collection tube.
7. Add 600µL of rinse solution
PW to the adsorption column CP3 (please check whether absolute ethanol has been
added first), centrifuge at 12,000 rpm (~13,400xg) for 30-60 sec, pour off the
waste liquid in the collection tube, and put the adsorption column CP3 into the
collection tube.
8.
Repeat step seven.
9. Put the adsorption column CP3 into the collection tube
and centrifuge at 12,000 rpm (13,00xg) for 2 minutes to remove the residual
washing liquid in the adsorption column. (The residual ethanol in the rinsing
solution will affect the subsequent enzyme reaction experiments. In order to
ensure that downstream experiments will not be affected by residual ethanol, it
is recommended to remove the cap of the
adsorption column CP3 and place it at room
temperature for several minutes to dry the residual rinse solution in
the adsorption material completely).
10. Put the adsorption column CP3 in a clean centrifuge
tube, add 50-100℃L elution buffer H2O dropwise to the middle part of the adsorption membrane, place℃
at room temperature for 2 minutes, and centrifuge at 12,000 rpm (-13,400Xg) for
2 minutes to remove the plasmid The solution was collected into a centrifuge
tube.
Experiment 2
Experimental purpose:to construct the
mutant form of NAMPT selected by directed evolution.
Method:Site-directed mutagenesis
STEP 1 - Primer Design
Precautions
1. The primer length should be generally between 15-30
bases. Otherwise, it will not be conducive to the reaction of the polymerase.
1) Primers that are too short will reduce amplification
specificity.
2) On the other hand, if the primer is too long, the
annealing temperature will increase.
2. The GC content of the primer should be between 40% and
60%, and the Tm value is preferably close to 72℃.
1) Tm value: The Tm value (melting temperature) of the
primer = 4(G+C)+2(A+T), try to ensure that the Tm value of the upstream and
downstream primers is consistent, generally not exceeding 2℃.
3. The bases are supposed to be distributed randomly.
1) Avoid repeating nucleobases to cause false priming;
try to avoid complementarity between primers to prevent the formation of primer
dimers or hairpin structures.
4. Primer specificity: If the 3' end of the primer
contains a G or C residue, the specificity of the primer can be increased.
STEP 2 - PCR Reaction
1.
Reaction mix
|
Reagent |
Volume ( µL) |
|
PCR grade water |
7 |
|
KOD One™ PCR Master Mix |
10 |
|
10 µM Primers F/R |
1 each |
|
Template |
1 |
|
Total volume |
20 |
2.
PCR cycle
|
Process |
Temperature (℃) |
Time(s) |
Cycle times |
|
Pre-denaturation |
98 |
180 |
1 |
|
denaturation |
98 |
10 |
22 |
|
Annealing |
58 |
5 |
|
|
Elongation |
68 |
90 |
|
|
Elongation 2 |
65 |
300 |
1 |
|
preservation |
4 |
- |
1 |
STEP 3 - Prepare Nucleic Acid Gel
1. Weigh 0.3g of agarose powder, add it to 30ml TAE
solution, and heat it in a microwave oven to fully melt the agarose powder
until the solution is clear and transparent.
2. Pour the agarose gel solution into the gel tank and
insert the comb.
3. After about 30 min of solidification, take out the
nucleic acid gel and use it immediately or store it in TAE solution for later
use.
STEP 4 - Nucleic acid gel electrophoresis
Take 10 µL of the PCR product
for gel identification, and check whether there is a single band at the
position corresponding to the size of the band, so as to infer whether the PCR
is successful.
STEP 5 - Add the Dpnl enzyme digestion template
1. Reaction mix
|
Reagent |
Volume ( µL) |
|
water |
7 |
|
10 µL FlashOne™ buffer |
2 |
|
DNA |
10 |
|
FlashCut™
Dpnl |
1 |
|
Total volume |
20 |
2.
Operating process
1) Preparation of reaction system.
2) Gently suck or flick the tube wall to mix well (do not
vortex), and then centrifuge instantaneously to collect the wall-hanging
droplets.
3) Incubate at 37℃ for 60min.
STEP 6 - Transform
1. Take 50 µL of competent cells melted on an ice bath,
add the DNA of interest, mix gently, and leave in an ice bath for 30 min.
2. Heat in a water bath at 42 ℃ for 45 sec, then quickly
transfer the tubes to an ice bath for 2 min, the process without shaking the
centrifuge tubes.
3. Add 500 µL of sterile LB medium (without antibiotics)
to each centrifuge tube, mix well and place in 1 h aphine plates at 37 ℃, 200
rpm to revive the bacteria.
4. Pipette different volumes of transformed competent
cells onto LB agar medium containing the appropriate antibiotics, and spread
the cells evenly. Place the plate at 37 ℃ until the liquid is absorbed, invert
the plate and incubate at 37 ℃ overnight.
STEP 7 - Pick multiple single clones for sequencing
identification
1. Each
person picked 3 colonies, cultured them into a medium containing ampicillin
antibiotic, and incubated them at 37℃ over night.