DESIGN
Protocol
Configure 2× FastPfu Fly Mix, according to the following system for subsequent PCR system:
The PCR system for gel-recovered fragments is as follows:
The FastPfu Fly DNA Polymerase reaction procedure is as follows:
The colony PCR system is as follows:
The reaction procedure of 2×Taq DNA Polymerase is as follows:
After the colony PCR system is set up, take 0.5 µL points of the mixed system on the corresponding
antibiotic-resistant replica for subsequent inoculation of plasmids and bacterial species
preservation.
Pipette Tips Recycling Steps
After configured the corresponding system
of 1% of Agarose gel, adding 10,000 diluted fold nucleic acid dye SYBR Safe, and then
expanding PCR product to 140V voltage for running gel, and finally fragment was retained by
gel extraction kit which is selected from Beijing the gel recovery kit of Tiangen
Biochemical Technology Co., Ltd. has been optimized according to experimental requirements.
The specific operation following steps are:
Preparation of yellow pipette tip adsorption
column CA2: Sterilize the tweezers with 75% alcohol, take out the blue fixing ring in
the adsorption column CA2 of the glue recovery kit, taking out the filter membrane in the
adsorption column, and use tweezers to take half of each into the yellow gun head, plug the
filter membrane as tightly as possible.
1.
Column equilibration steps: put the yellow pipette tip
adsorption column CA2 in a new tube, adding 200 µL balanced solution BL to it,
centrifuging at 12000 rpm for 1 min, discard the waste liquid in the collection tube,
and put the adsorption column back into the middle of collection tube.
2.
Cut a single target DNA band from the agarose gel and put
it into a 1.5 mL clean centrifuge tube.
3.
Add 150-250 µL of solution PN depending on the size of
the cut gel block (if it is not clear whether the gel block is small, adding 200 µl),
and place it in a metal bath at 65°C for 5-10 minutes. During this time, gently turn the
centrifuge tube up and down until the gel block completely dissolved. Note: At this
time, place the sterile dd H2O on a metal bath at 65°C for preheating.
Note: For the recycle of small fragments <300bp, you can add 1/2 the volume of the gel block
(usually 100 µL) of isopropanol after adding PN to complete the sol to increase
the DNA recovery rate; after the gel block is completely dissolved, please lower the
temperature to Load the column at room temperature because the adsorption column has a
strong ability to bind DNA at room temperature.
4.
Add 200 µL of the solution obtained in the previous
step to the yellow pipette tip adsorption column CA2 after column equilibration (the
adsorption column is placed in the collection tube), mark the yellow pipette tip, place
it at room temperature for 2 minutes, and centrifuge at 12000 rpm for 1 minute. Pour off
the waste liquid in the collection tube, and put the yellow pipette tip adsorption
column CA2 back into the collection tube. Then add the remaining solution to the yellow
pipette tip adsorption column CA2 without placing it, and centrifuge at
12000rpm for 1min.
5.
Add 200µL of rinsing solution PW to the yellow pipette
tip adsorption column CA2 (please check whether absolute ethanol has been added before
using), centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection
tube, and put the yellow pipette tip adsorption column CA2 back into the collection tube
middle.
6.
Repeat step 5.
7.
Put the yellow tip adsorption column CA2 back into the
collection tube, 12000 rpm air from 2min, try a divisible rinse of the PW. Take an
appropriate amount of 1.5mL centrifuge tube, put the yellow pipette tip adsorption
column CA2 in a new 1.5mL centrifuge tube, and place it in a metal bath at 65°C for 5
minutes to thoroughly dry the rinsing liquid PW to prevent residual rinsing liquid from
affecting the next experiment.
8.
Take 65 ℃ preheated dd H2O around 16-20μL, was added
dropwise to a central membrane adsorption, placing at room temperature for 2min, 12000
rpm centrifugation for 2min; the collected DNA was added back to the centrifugal
adsorption column, placed 2min, 12000 rpm centrifugation for 2min, Collect DNA.
Plasmid extraction
Use the plasmid small extraction kit of Beijing Tiangen
Biochemical Technology Co., Ltd. to extract plasmids from the bacteria cultured overnight.
The specific operation steps are as follows:
1.
Centrifuge 5 mL of the bacterial solution cultured
overnight at 4000 rpm for 8-10 min to collect the bacterial cells, and remove the
supernatant as much as possible.
2.
Was added to the bacterial pellet leaving bacteria shake
tube 150 µL solution Pl (previously added RNAse and TIANRed), by pipetting or vortexing
(the degree of mixing of the cells on the extraction and subsequent cleavage of the
plasmid Quantity and purity), and transfer it from the 14 mL shaking tube to a new 1.5
mL EP tube.
Note: The addition of TIANRed reagent has no effect on
post- PCR, enzyme digestion and sequencing. After thoroughly inverting and mixing, the
solution is clear red. Add the mixed solution to the collected bacteria and mix thoroughly.
Due to the presence of the bacteria, the mixed solution is turbid red.
3.
Add 150 µL P2 buffer to a 1.5 mL EP tube, and gently mix
upside down 7-8 times to ensure that the bacteria are fully lysed.
Note: At this time, the bacterial solution should become
clear and viscous. If it does not become clear, it may be due to too much bacteria and
incomplete lysis. The amount of bacteria should be reduced (do not shake vigorously,
otherwise it will interrupt the genomic DNA and cause the extracted plasmid If there
are DNA fragments, the operation time of this step should not exceed 5 min to avoid damage
to the plasmid).
Due to the use of TIANRed, after adding solution P2 and
mixing thoroughly, the color of the solution is clear purple. If there is a turbid red in
the purple, it means that the lysis is not sufficient. Continue to mix until the color of
the solution completely changes to a clear purple.
4. Add 350 µL solution P5 buffer to the 1.5 mL EP tube,
gently invert and mix 12-20 times to ensure that the bacteria are fully mixed, at this time
flocculent precipitation will appear, then immediately centrifuge at 12000 rpm for 12 min.
Note: After P5 is added, it should be inverted and mixed
immediately to avoid local micro-precipitation. The supernatant containing a small amount of
small white precipitate has no effect on the subsequent test. If the supernatant is still
mixed with micro-precipitate, it can be centrifuged again and there will be no subsequent
experiment. Influence. If there are a lot of tiny white precipitates in the supernatant,
centrifuge again and take the supernatant.
Due to the use of TIANRed, after adding solution P5 and
mixing thoroughly, the solution is clear yellow. If purple is mixed with yellow, it means
that the renaturation is insufficient. Continue to mix until the color of the solution is
completely clear yellow.
5. Use a pipette to transfer the supernatant collected in the
previous step to the CP3 adsorption column (the adsorption column is placed in the
collection tube), try not to absorb the precipitate. Centrifuge at 12000 rpm for 1 min,
discard the waste liquid in the collection tube, and put CP3 back into the collection tube.
6.Add 400 µL of PWT washing solution to the adsorption
column CP3 (confirm whether absolute ethanol is added in advance), centrifuge at 12000
rpm for 1 min, discard the waste liquid in the collection tube, and put CP3 back into the
collection tube.
7.Repeat the operation (6).
8. Put the CP3 back into the collection tube, and centrifuge
the empty tube again at 12000 rpm for 2 minutes to ensure that the residual washing solution
on the CP3 adsorption membrane is completely removed. Then let it stand for 5 min in a metal
bath at 60°C to completely dry the remaining washing liquid on the adsorption membrane.
9. Put CP3 into a new clean 1.5 mL EP tube, add 50-60 µL of
sterilized dd H
2
O to the center of the CP3 adsorption membrane ( preheat
at 65°C in advance to improve the elution efficiency), and let stand at room
temperature 2 After min , centrifuge at 12000 rpm for 2 min to collect the plasmid in
the tube (if you want to improve the elution efficiency, you can aspirate the plasmid in
the collection tube and add it to the center of the adsorption membrane again,
and centrifuge at 12000 rpm for 2 min to enrich the plasmid again). The plasmid should
be used immediately, otherwise it should be stored at -20°C to prevent DNA degradation.
Gibson Assembly
The Gibson Assembly system is as follows：
According to the above system, the configuration 10 µL
of Gibson assembly system at 60℃ the reaction 1h to complete the assembly clip, and then
Escherichia coli DH5α transformed.
Golden Gate Assembly
The Golden Gate Assembly system is as
follows:
①
If the number of inserts is 1~10 ,
use 420 units ; if the number of inserts is more than 10 , use 630units;
②
Available endonuclease: BsmBI-v2(NEB #R0739, 10U/µl), BsaI-HFv2 ( NEB #R3733S
20U/µl); If the number of inserts is 1~10 , use 24 units ; if the number of
inserts is greater than 10 , use 36 units;
③
The amount of each
insert can be based on the amount of vector added and the vector and the
length of the insert.
The Golden Gate Assembly reaction procedure is as follows:
According to the above reaction system and program configuration 20µL of the Golden Gate assembly
system, after completion of the reaction for E. coli DH5α transformed.
The enzyme linkage system is as follows:
Table 9 T4 DNA Ligase enzyme linkage system
According to the above system, configure a 10 µL ligation
product, incubate at 20°C for 4 h to complete the construction of the ligation product, and
then transform E. coli DH5α.
Escherichia coli
competent transformation
1. Take out the prepared competent state in the ultra-low
temperature refrigerator at -80 ℃ and thaw it on ice. After the defrosting is complete,
add 10 µL reaction product (add enzyme or assembly product immediately after thawing to
avoid affecting the transformation quality of competent state). Flick the tube wall with
fingers to mix the bacteria, and bath in ice for 20-30 min.
2. After the ice bath is completed, heat shock in a 42 ℃
water bath for 45-90 seconds, and immediately let it stand on ice for 5 min .
3. Add 400 µL SOC liquid medium and resuscitate at 220 rpm in
a shaker at 37 ℃ for 1 h.
4. Spread all the recovered bacterial liquid on the
corresponding resistant LB solid medium, and invert it overnight in an incubator at 37 °C.
Yeast Competent Preparation
1. Spread the stored yeast on YPD solid medium
and incubate at 30 °C for 2 days . Pick a single colony was inoculated into YPD liquid
medium at 30 shaker in ℃ 230rpm cultures 18-20 h .
2. Re-inoculate the overnight cultured yeast into 10mL
YPD medium so that the initial OD600 is 0.1-0.15; culture it in a shaker at 30°C, 230
rpm to OD600 = 0.4~0.6, and collect the bacteria by centrifugation at 4000 rpm for 5
min body;
3. Resuspend with 1mL sterile water, centrifuge
at 4000rpm, 5min to remove the supernatant; add 1mL cell suspension (TE/LiAc/H2O=1:1:8)
at 4000rpm, 5min, centrifuge again to remove the supernatant;
4. Gently resuspend the bacteria with 150 µL cell suspension
to prepare yeast competent cells.
Yeast assembly and transformation
1. Boil the single-stranded salmon sperm DNA (10mg/mL) in a
boiling water bath for 10 minutes before use, and then use an ice bath for
later use. Add 20 µL to the 300 µL conversion solution (TE/ LiAc/ PEG3350 =1:1:8) Mix
gently.
2. Take the purified DNA fragments and ensure that the final
concentration of the added fragments is ≈200 ng, then the final concentration of the
linearized vector is ≈50 ng (4: 1), and the fragments are in an equimolar ratio. Mix, add to
the yeast competent cells, flick and mix (to ensure optimal transformation efficiency, the
total volume of the fragment mixture should not exceed 60 µL, within this transformation
volume range, the concentration of the mixture can be expanded to ensure higher Assembly
efficiency).
3. Add the prepared yeast transformation solution to the
yeast competent cells (the yeast transformation solution is configured according to the
number of transformations), and vortex at low frequency to mix.
4. Incubate at 30°C (water bath, metal bath
or 30°C incubator) for 45 min.
5. Then place it in a 42 ℃ water bath for 15 minutes, ice
bath for 2 minutes, and centrifuge at 4000 rpm for 5 minutes to remove the
supernatant, resuspend and wash the bacteria with 600µL ddH
2
O, then centrifuge at 4000 rpm for 5 minutes to remove
the supernatant again, and add 120µL ddH
2
O to resuspend the bacteria, spread on
the SC-U auxotrophic solid plate, incubate at 30 ℃ for 3 days, select recombinants
for verification
Reagents:
1. YPD medium ( 1L ) : 10g yeast extract, 20g peptone
dissolved in 900 mL deionized water at 121 ℃ for 20 minutes , add 100mL 20% glucose mother
liquor; add 15g agar powder before sterilization, it is a solid agar medium.
2. 20% glucose mother liquor: Weigh 20g glucose
in 100mL deionized water, sterilize it at 115 ℃ for 15 minutes , and store it at 4 ℃ for
later use .
3. SC-Ura Defective Medium ( 1L ) : Weigh 6.7g Yeast
Synthetic Drop-out Medium without uracil , take 1.3g Yeast Synthetic Drop-out Medium without
uracil , dissolve in 900mL deionized water, sterilize at 121 ℃ for 20 minutes , add 100 mL
20% glucose or galactose mother liquor is used as the carbon source; 15g agar powder
is added before sterilization to form a solid agar medium.
4. 10 × LiAc solution: Weigh 8.16g LiAc • 2H
2
O solid, dissolve it in 80 mL deionized water, adjust
the pH to 7.5 with HAc, add deionized water to make the volume to 100 mL, obtain 0.8M
LiAc solution and quench Bacteria spare.
5. 10 × TE solution: 1.211g Tris, 0.37g EDTA dissolved in 80
mL deionized water, adjust the pH to 7.5 with HCl, add deionized water to make the volume
to 100 mL, obtain 0.1M Tris-HCl, 10mM EDTA sterilization spare.
6. 50% PEG3350: Weigh 25g of PEG3350 solid and dilute
to 50ml with deionized water, filter and sterilize 0.22 µm for use.
Yeast Recombinant Screening
After the yeast colony grows to a certain size (2 mm~3 mm),
pick the yeast transformants into 10 µL sterilized dd H2O water and mix well, stir and mix,
and take 4 µL dots in the auxotrophic type of SC-Ura On the medium plate, the adapter plate
style is shown in the figure below, and the colony PCR is initially screened after overnight
culture at 30°C.
Due to the thicker cell walls of Saccharomyces cerevisiae, it
is necessary to break the walls before doing the yeast colony PCR. Use a pipette tip to dip
the yeast colony and resuspend in 10µL 20mM sterile NaOH, and rupture the yeast cell wall in
a PCR machine according to the procedure in Table 10:
Take out the processed bacterial solution from
the PCR machine, mix it and take 1 µL as a template, use Taq DNA
Polymerase to verify the bacterial solution PCR according to the PCR reaction system
in Table 11 and the PCR program in Table 5, and screen the correct yeast recombinant:
