HCl, NaCl, chloroform, isopropanol, ethanol, LB Broth Agar Medium
Special medium
0.1% FBS :
· 45mL DMEM
· 45uL Fetal Bovine Serum
Protocol:
Target Guide Sequence Cloning protocol
In order to clone the target sequence into the lentiCRISPRv2 or lentiGuide-Puro backbone, synthesize two oligos of the following
form.
Lentiviral vector digestion, oligo annealing and cloning into digested vector: 1. Digest and dephosphorylate 5ug of the lentiviral CRISPR plasmid with BsmBI for
30 min at 37C: 5 ug lentiCRISPRv2 or lentiGuide-Puro
3 ul FastDigest BsmBI (Fermentas)
3 ul FastAP (Fermentas)
6 ul 10X FastDigest Buffer
0.6 ul 100 mM DTT (freshly prepared)
X ul ddH2O
60 ul total
2. Gel purify digested plasmid using QIAquick Gel Extraction Kit and elute in EB. If BsmBI digested, a ~2kb filler piece should be present on the gel. Only gel purify
the larger band. Leave the 2kb band.
3. Phosphorylate and anneal each pair of oligos: 1 ul Oligo 1 (100 μM)
1 ul Oligo 2 (100 μM)
1 ul 10X T4 Ligation Buffer (NEB)
6.5 ul ddH2O
0.5 ul T4 PNK (NEB M0201S)
10 ul total
Use the T4 Ligation Buffer since the buffer supplied with the T4 PNK enzyme does
not include ATP (or supplement to 1mM ATP).
Put the phosphorylation/annealing reaction in a thermoccler using the following
parameters:
37℃ 30 min →95℃ 5 min and then ramp down to 25℃ at 5℃/min
4. Dilute annealed oligos from Step 3 at a 1:200 dilution into sterile water or EB. 5. Set up ligation reaction and incubate at room temperature for 10 min: X ul BsmBI digested plasmid from Step 2 (50ng)
1 ul diluted oligo duplex from Step 4
5 ul 2X Quick Ligase Buffer (NEB)
X ul ddH2O
10 ul subtotal
1 ul Quick Ligase (NEB M2200S)
11 ul total
Also perform a negative control ligation (vector-only with water in place of oligos)
and transformation.
6. Transformation into DH5α bacteria.
Extract human genome DNA:
1. Cultured Cell Preparation a. Harvest cells
• Attached cell cultures: Release cells with trypsin. Pellet up to 5 × 106 cells
for 5 minutes at 300 × g; remove the culture medium completely and discard. Continue
to step 2a.
• Suspension cell cultures: Pellet up to 5 × 106 cells for 5 minutes at 300 × g;
remove the culture medium completely and discard. Continue with step 2a.
Note: Cells can be harvested, aliquotted into 1.5 mL micocentrifuge tubes and flash-frozen
in liquid nitrogen, then stored at –70 °C for several months before preparing DNA.
b. Resuspend cells
Resuspend the pellet thoroughly in 200 µL of Resuspension Solution. If previously
frozen, allow the cell pellet to thaw slightly before resuspending. If residual RNA
is not a concern, continue with step 3a.
Optional RNase A treatment: If RNA-free genomic DNA is required, add 20 µL of RNase
A Solution and incubate for 2 minutes at room temperature, then continue with step
3a.
c. Lyse cells
Add 20 µL of the Proteinase K solution to the sample, followed by 200 µL of Lysis
Solution C (B8803). Vortex thoroughly (about 15 seconds), and incubate at 70 °C for
10 minutes. A homogeneous mixture is essential for efficient lysis. Continue with
step 4.
DNA Isolation From All Listed Sample Types
2. Column preparation Add 500 µL of the Column Preparation Solution to each pre-assembled GenElute Miniprep
Binding Column (with a red o-ring, not to be confused with other GenElute kits) and
centrifuge at 12,000 × g for 1 minute. Discard flowthrough liquid.
Note: The Column Preparation Solution maximizes binding of DNA to the membrane resulting
in more consistent yields.
3. Prepare for binding Add 200 µL of ethanol (95–100%) to the lysate; mix thoroughly by vortexing 5–10
seconds. A homogeneous solution is essential.
4. Load lysate Transfer the entire contents of the tube into the treated binding column from step
4. Use a wide bore pipette tip to reduce shearing the DNA when transferring contents
into the binding column. Centrifuge at ≥6500 × g for 1 minute. Discard the collection
tube containing the flowthrough liquid and place the binding column in a new 2 mL
collection tube.
5. First wash Prior to first use, dilute the Wash Solution Concentrate with ethanol as described
under Preparation Instructions. Add 500 µL of Wash Solution to the binding column
and centrifuge for 1 minute at ≥6,500 × g. Discard the collection tube containing
the flow-through liquid and place the binding column in a new 2 mL collection tube.
6. Second wash Add another 500 µL of Wash Solution to the binding column; centrifuge for 3 minutes
at maximum speed (12,000-16,000 × g) to dry the binding column. The binding column
must be free of ethanol before eluting the DNA. Centrifuge the column for one additional
minute at maximum speed if residual ethanol is seen. You may empty and re-use the
collection tube if you need this additional centrifugation step. Finally, discard
the collection tube containing the flowthrough liquid and place the binding column
in a new 2 mL collection tube.
7. Elute DNA Pipette 200 µL of the Elution Solution directly into the center of the binding column;
centrifuge for 1 minute at ≥6,500 × g to elute the DNA. To increase the elution efficiency,
incubate for 5 minutes at room temperature after adding the Elution Solution, then
centrifuge.
Optional: A second elution can be collected by repeating step 9 with an additional
200 µL of Elution Solution and eluting into a new 2 mL collection tube (provided)
or into the same 2 mL collection tube as used for the first eluate.
The eluate contains pure genomic DNA. For short term storage of DNA, 2–8 °C is recommended.
For long-term storage of DNA, -20 °C is recommended. Avoid freezing and thawing,
which causes breaks in the DNA strand. The Elution Solution will help stabilize the
DNA at these temperatures.
Standard PCR Protocol:
1. Add DNA template up to 50ng into PCR tube,(x ul).
2. Add reagents as follow:
· dNTP: 4ul
· 10x Dream Taq Buffer: 5ul
· Primer: 1ul (after diluted)
· Dream Taq : 0.25ul
· ddH2O: (33.5-x)ul
· MgCl2: 4ul
· Template:
<500ng Total: 50ul
Mix gently by vortex and briefly centrifuge to collect all components to the
bottom of the tube.
3. PCR pocedure:
98℃(2min)→ 98℃(10s)→55℃(5s)→72℃(1Kb/min)→72℃(5min)→4℃(∞) ,25 cycles
4. The amplification parameters will vary depending on the primers and the thermal
cycler used. It may be necessary to optimize the system for individual primers,
template, and thermal cycler.
PCR purification
Method:Cycle-Pure Kit(200)
1. Perform agarose gel/ethidium bromide electrophoresis to analyze PCR product.
2. Determine the volume of the PCR reaction. Transfer the sample into a clean 1.5ml
micocentrifuge tube and add 4-5 volumes of Buffer CP. For PCR products smaller than
200bp, add 6 volumes of Buffer CP.
3. Vortex thoroughly to mix. Briefly spin the tube to collect any drops from the
inside of the lid.
4. Place a HiBind DNA Mini Column into a provided 2 ml collection tube.
5. Add the mixed sample from step 3 to the HiBind DNA Mini Column and centrifuge
at 13,000 x g for 1 minute at room temperature. Discard the flow-through liquid and
place the HiBind DNA Mini Column back into the same collection tube.
6. Add 700µl of DNA Wash Buffer and centrifuge at 13,000 x g for 1 minute. Discard
the flow-through liquid and place the HiBind DNA Mini Column back into the same collection
tube.
7. Add 500µl of DNA Wash Buffer and centrifuge at 13,000 x g for 1 minute. Discard
the flow-through liquid and place the HiBind DNA Mini Column back into the same collection
tube.
8. Centrifuge the empty HiBind DNA Mini column for 2 min at maximal speed ( ≥13,000
x g ) to dry the column matrix.
9. Place the HiBind DNA Mini column into a clean 1.5ml micocentrifuge tube. Depending
on the desire concentration of the final product, add 30-50 µl of Elution Buffer
(10mM Tris, pH8.5) or water directly onto the center of column matrix.
Restriction endonuclease digestion
Gel Extraction
Method:QIAquick Gel Extraction Kit I(250)
1. Excise the gel slice containing the DNA band with a clean, sharp scalpel. Minimize
the size of the gel slice by removing excess polyacrylamide.
2. Weigh the gel slice. Add 1–2 volumes of diffusion buffer to 1 volume of gel(i.e.,
100–200 µl for each 100 mg of gel).
3. Incubate at 50°C for 30 min.
4. Centrifuge the sample for 1 min.
5. Carefully remove the supernatant using a pipet or a drawn-out Pasteur pipet.
Pass thesupernatant through a disposable plastic column or a syringe containing either
aWhatman GF/C filter or packed, siliconized glass wool to remove any residual polyacrylamide.
6. Determine the volume of the recovered supernatant.
7. Add 3 volumes of Buffer QG to 1 volume of supernatant and mix. Check that the color
of the mixture is yellow.
8. Place a QIAquick Spin Column in a provided 2 ml collection tube.
9. To bind DNA, apply the sample to the QIAquick Spin Column and centrifuge for30–60 s.
10. Discard flow-through and place QIAquick Spin Column back into the same collection
tube.
11. To wash, add 0.75 ml Buffer PE to column and centrifuge for 30–60 s.
12. Discard flow-through and place QIAquick Spin Column back in the same tube.
Centrifuge column for an additional 1 min at maximum speed.
13. Place QIAquick Spin Column into a clean 1.5 ml micocentrifuge tube.
14. To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the
QIAquick Spin Column and centrifuge for 1 min.
Construct expression vector
Make the following reaction system.
10×ligation buffer 2 u l
Vector 50 ng
Insert the DNA fragments (and the molar ratio of carrier DNA is about 3) * 2
T4 DNA Ligase (350 U/u l) 1 u l
Sterilization of water
Total 20 u l
Let the connecting system responses at 16 ℃ in 4 hours.
Use part of the connection liquid for transformation. Remain can be preserved in
- 20 ℃.
E.coli Competent cell:
1. Innoculate a single colony of E. coli into 5 ml LB and grow O/N at 37
2. Innoculate 1 ml into 100ml and grow to an O.D. 600 of 0.4
3. Aliquot the culture into 2x 50ml pre-chilled Sorvall tubes and leave on ice for
5 -10 mins
4. Centrifuge cells for 7 mins at 3000rpm, 4℃ without brakes
5. Pour off supernatant and resuspend each pellet in 10 ml of ice-cold CaCl2 soln
6. Centrifuge cells for 5 mins at 2500rpm, 4 degree. Discard supernatant and resuspend
each pellet in 10 ml of cold CaCl2 solution.
7. Keep resuspended cells on ice for 30min.
8. Centrifuge cells for 5 mins at 2500rpm, 4℃. Discard supernatant and resuspend
each pellet in 2ml of ice-cold CaCl2.
9. Dispense cells into pre-chilled sterile eppendorfs.
10. Freeze immediately at -70 ℃
Notice: CaCl2 Soln: 60mM CaCl2, 15% Glycerol
Transformation of competent cells
Melt the cells in the ice.
2. Add the DNA which need to be transformed to DH5 alpha cells.Pay attention to
the purpose of DNA volume no more than one over ten of the cells suspension liquid
product, gently rotating centrifugal tube with blending contents, ice-bath place
for 30 minutes.
3. Put the centrifugal tube at 42 ℃ water bath for 60-90 seconds, and then quickly
transmitted it to the ice -bath place for 2-3 minutes, be careful not to shake the
centrifugal tube.
4. Add 1mL LB culture medium without antibiotic in centrifuge tube, 37 ℃ 220 rpm
oscillating culturing for 1 hour.
5. Centrifuge and suck out the culture, suspending bacteria and coating on the plate.
Coating residual bacteria liquid can be retained 4 ℃, if the number of colonies
too little, can put the rest of the bacteria liquid coating in a new plate again.
Plasmid Extraction:
Method: High-yeild Plasmid DNA MiniPreparation Kit(200)
1. Isolate a single colony from a freshly streaked selective plate, and innoculate
a culture of 1- 5 mL LB medium containing the appropriate selective antibiotic. Incubate
for 12-16 hours at 37°C with vigorous shaking (300 rpm). Use a 10-20 mL culture tube
or a flask with a volume of at least 4 times the volume of the culture.
2. Centrifuge at 10,000 x g for 1 minute at room temperature. Decant or aspirate
and discard the culture media.
3. Add 250 µL Solution I/RNase A. Vortex or pipet up and down to mix thoroughly.
Complete resuspension of cell pellet is vital for obtaining good yields.
4. Add 350 µL Solution III. Immediately invert several times until a flocculent
white precipitate forms.
5. Centrifuge at maximum speed (≥13,000 x g) for 10 minutes. A compact white pellet
will form. Promptly proceed to the next step.
6. Insert a HiBind® DNA Mini Column into a 2 mL Collection Tube.
7. Transfer the cleared supernatant from Step 5 by CAREFULLY aspirating it into
the HiBind® DNA Mini Column. Be careful not to disturb the pellet and that no cellular
debris is transferred to the HiBind® DNA Mini Column.
8. Centrifuge at 10000g speed for 1 minute.
9. Discard the filtrate and reuse the collection tube.
10. Add 500 µL HB Buffer.
11. Centrifuge at 10000g speed for 1 min.
12. Discard the filtrate and reuse collection tube.
13. Add 700 µL DNA Wash Buffer.
14. Centrifuge at 10000g for 1 minute.
15. Discard the filtrate and reuse the collection tube.
16. Centrifuge the empty HiBind® DNA Mini Column for2 min at 13000g to dry the column
matrix.
17. Transfer the HiBind® DNA Mini Column to a clean 1.5 mL micocentrifuge tube.
18. Add 30-50 μL Elution Buffer or sterile deionized water directly to the center
of the column membrane.
19. Let sit at room temperature for 5 minute.
20. Centrifuge at 13000g for 1 minute.
21. Store DNA at -20°
HEK 293 Cells transfection
Use this pocedure to transfect plasmid DNA into HEK 293 cells in a 24-well format (for other formats, see Scaling Up or Down
Transfections, below). All amounts and volumes are given on a per well basis.
1. The day before transfection, trypsinize and count the cells. Plate 0.5 -1.25x105
cells per well in 0.5 ml of complete growth medium. Cell density should be 50-80%
confluent on the day of transfection.
2. (Optional) The day of transfection, remove growth medium from cells and replace
with 0.5 ml of complete growth medium.
3. For each well of cells to be transfected, dilute 0.5 μg of DNA in 100 μl of Opti-MEM®I
Reduced Serum Media without serum.
4. For each well of cells, add 0.75-1.75 μl of Lipofectamine LTX™Reagent into the
above diluted Opti-MEM®:DNA solution, mix gently and incubate 30 minutes at room
temperature to form DNA- Lipofectamine LTX™Reagent complexes.
5. After 30 minute incubation, add 100 μl of the DNA- Lipofectamine LTX™Reagent
complexes directly to each well containing cells and mix gently by r℃king the plate
back and forth.
6. Complexes do not have to be removed following transfection. Incubate the cells
at 37℃ in a CO2 incubator for 18-24 hours post-transfection before assaying for transgene
expression.
Lentivirus Infection
1. Seed cells at appropriate density in 6 ml in 6 cm plates.
a Adherent cells: seed 1 day prior to infection.
b Suspension cells: seed day of infection in media containing polybrene.
2. Add virus to cells:
(Adherent cells): Remove growth media and add fresh media containing polybrene.
Alternatively, remove a portion of the growth media and supplement with media containing
polybrene. Adjust volumes and polybrene concentration to achieve the correct final
polybrene concentration (8 μg/ml).
3. Viral infection:
Incubate cells overnight.
Change media 24 h post-infection. Remove media and replace with 6 ml fresh growth
media. If antibiotics selection is desired, use fresh growth media containing antibiotics.
Note: Puromycin concentration should be optimized for each cell line; typical concentrations
range from 2-5 μg/ml.
4. Incubate cells, replacing growth media (with antibiotics, if desired) as needed
every few days. Incubation periods are highly dependent on the post-infection assay.
Note: All lentiviral pocedures should be carried out in accordance with biosafety
requirements of the host institution.
Extract the cell RNA:
1. Lyse cells
Precipitate cells by centrifugation. In the RL reagent, repeatedly blow the lysate
with the pipette gun.
2. Mix homogenized samples violently and incubate for 5 minutes at 15-30℃ for the
complete decomposition of nucleoprotein.
3.Under the condition of 4℃, 12000rpm centrifugefor 10 minutes, transfer the supernatant
carefully into a new RNase-free tube.
4. Add 0.2ml chloroform each 1ml RL. Shaking violently for 15 seconds and incubate
it for 3 minutes at room temperature.
5. Centrifugate 12,000rpm at 4℃ for 10 minutes, RNA exists in the upper aqueous
phase. Transfer the aqueous phase to new tube.
6. Adding 1 times volume of 70% ethanol into the adsorption column RA.
7. Centrifuge 45 seconds at 10, 000rpm, discard the waste, reset the adsorption
column to recovery tube.
8. Add 500 uL protein solution RE, 12, 000rpm centrifugation for 45 seconds, discard
waste liquid.
9. Add 700 ul washing solution RW, 12000 rpm centrifugation for 60 seconds, discard
waste liquid.
10. Add 500 uL RW, 12000 rpm centrifugation for 60 seconds, discard waste liquid.
Then empty centrifuge1min to remove ethanol residue as much as possible.
11. Add digestive fluid to RA adsorption column at 42℃ water-bath for 5min and then
remove it, repeat the steps 8 and 9 then step 12.
12. Put the adsorption column RA back into the collection tube, centrifuge at 12,000
rpm for 2 minutes.
13. Remove the adsorption column RA into a RNase-free centrifugation tube, according
to the expected product of RNA add 50-80 uL RNase-free water in the middle part of
the adsorption membrane.Set at room temperature for 2 minutes, 12,000 rpm centrifugation
for 1 minutes.store eluted RNA at -70℃.
RT-PCR
Method: GoScript Reverse Transcription System
1. micocentrifuge the reagents of kit
2. Prepare mix A as follows:
· RNA: x ul ,make sure the RNA content is up to 1ug/ul.
· oligdT: 1 ul
· dNTP: 1 ul
· DEPC water: (8-x) ul
· Total: 10 ul
3. Incubate mix A for 5 minutes at 70℃ in PCR machine. Then, set on the ice for
5min.
4. Prepare mix B as follows:
· MgCl2: 2ul
· DEPC water: 1.6ul
· GoScript: 4ul (5x Reaction Buffer)
· NucMix: 1ul
· RNase: 0.4ul
· RTE: 1ul
· Total: 10ul
5. Add mix B to mix A. Set PCR pocedure:
25℃ 5minutes → 42℃ 60minutes → 70℃ 15minutes → 4℃ ∞
Insert PCR tube with mixed system into PCR machine, store at -20℃。
Real-time PCR
Method: SYBR qPCR Quick protocol
Standards · Dilute the cDNA ~4-fold (e.g. 21μl sample + 59μl H2 O = 80μl)
· Pool an appropriate amount from each sample to create standard 1
· Create standards as follows
Pool 360μl standard 1 (25600)
1:4 90μl standard 1 + 270μl H2 O (6400)
· Mix well at each step.
Plate · For the No cDNA wells, pipette 8μl of H2O.
· For the standard wells, pipette 8μl of standard.
· For the sample wells, pipette 8μl of sample.
· Run each sample and standard in duplicate.
Assay Make 12μl of master mix for each well, plus some excess
1 Rxn: 10μl SYBR Green Mix 52 Rxn: 520μl
SYBR
0.4μl Forward Primer (10μM st℃k)
20.8μl F
0.4μl Reverse Primer (10μM st℃k)
20.8μl R
1.2μl H2 O
62.4μl H2 O
12μl per well (+ 8μl cDNA = 20μl final volume ) 624μl
Analysis By Stepone software
