Ⅰ.Dealing
with biobricks
1.1
Storage
1.
Dried
DNA: room temperature
2.
Resuspended DNA: -20℃
freezer
3.
The linearized plasmid backbones
(25ng/ul at 50ul) should be stored at 4C or lower
1.2
Usage
1.
With a pipette tip, punch a hole through
the foil cover into the corresponding well to the Biobrick™-standard part that
you want. Make sure you have properly oriented the
plate. We recommend that you do not
remove the foil cover, as it could lead to cross contamination between the
wells.
2.
3.
Add 10uL of diH2O (deionized water), the
resuspension will become red due to the cresol red dye used during
manufacturing.
4.
Pipette 1 or 2uL of the resuspended DNA transform into your desired
competent cells, plate bacteria with the appropriate antibiotic* and grow
overnight.
5.
Pick a single bacterial colony and
transfer it into LB medium. Incubate the culture for 18 hours with vigorous
agitation.
6.
Store the single colony in physiological
saline and on inclined plane, and make recognizable marks on the tube and on
the notebook.
Ⅱ. Preparation of
competent cells (Using Calcium Chloride)
2.1
Day 0:
Pick a single bacterial colony from a plate that has
been incubated for 16-20 hours at 37℃. Transfer the
colony into LB medium. Incubate the culture overnight (about 16 hours) at 37℃
with vigorous shaking.
2.2
Day 1:
1.
Transfer 1 ml of the culture into 100 ml
of LB medium. Incubate the culture for 2.5-3 hours at 37℃
with vigorous agitation (250-300rpm).
2.
Transfer 1.5 ml of the culture into
sterile ice-cold 1.5ml polypropylene tubes. Cool the cultures by storing the
tubes on ice for 10 minutes.
3.
Recover the cells by centrifugation at 2500
rpm for 5minutes at 4℃.
4.
Decant the medium from the cell pellets.
Resuspend each pellet by swirling or gentle vortexing in 100ml of ice-cold 0.1M
CaCl2 solution. Store the tubes on ice for 20minutes.
5.
Recover the cells by centrifugation at
2500 rpm for 5 minutes at 4℃.
6.
Decant the medium from the cell pellets.
Resuspend each pellet by swirling or gentle vortexing in 100ml of ice-cold 0.1M
CaCl2 solution.
7.
The suspension of competent cells should
be immediately used in transformation.
Attention:
[1]
Mix gently
[2]
The newly prepared competent cells
should be transformed immediately. They cannot be stored for long (except in
-80℃)
[3]
Keep sterile environment during
operation (sterilize the clean bench by ultra-violet, sterilize hands and implements
by alcohol).
[4]
Use heat-shock method to transform.
Ⅲ. Plasmid
Extraction（Using
Plasmid Mini Kit）
3.1
Things to do before starting:
•
Preheat Elution Buffer to 70°C if
Plasmid DNA is >10kb
•
Dilute DNA Wash Buffer with absolute
ethanol and Add vial of RNase A provided to Solution I.
3.2
Details:
1.
Isolate a single colony from a freshly
streaked selective plate, and inoculate a culture of 1- 5 ml LB medium
containing the appropriate selective antibiotic. Incubate for~ 12-16 hr at 37°C
with vigorous shaking (~ 300 rpm).
2.
Decant or Pellet bacterial cells by
centrifugation at 10,000 x g for 1 min at room temperature.
3.
Resuspend the bacterial pellet by adding
250 μl of Solution I/RNase A solution, and vortexing (or pipetting up and
down). Complete re-suspension (no visible cell clumps) of cell pellet is vital
for obtaining good yields. Transfer suspension into a new 1.5 ml
microcentrifuge tube.
4.
Add 250 μl of Solution II and gently mix
by inverting and rotating tube several times to obtain a clear lysate. A 2-3
minute incubation may be necessary. Avoid vigorous mixing as this will shear
chromosomal DNA and lower plasmid purity.
Note: Do not allow the lysis reaction to
proceed more than 5 min.
5.
Add 350 μl of Solution III and mix
immediately by inverting several times until a flocculent white precipitate
forms.
Note: It is vital that the solution is
mixed thoroughly and immediately after the addition of Solution III to avoid
localized precipitation.
6.
Centrifuge at 13,000 x g for 10 min at
room temperature. A compact white pellet will form. Promptly proceed to the
next step.
7.
Prepare a HiBind DNA Mini Column by
placing into a 2 mL collection tube.
8.
Add 100 μl of Equilibration Buffer.
Centrifuge at 13,000 x g for 30-60 seconds.
9.
Discard the flow-through liquid and
place the HiBind DNA Mini Column back into the same collection tube.
1)
Add the cleared supernatant from step 6
by CAREFULLY aspirating it into the HiBind DNA Mini Column. Ensure that the
pellet is not disturbed and that no cellular debris has carried over into the
HiBind DNA Mini Column. Centrifuge at 13,000 x g for 1 min at room temperature
to completely pass lysate through the HiBind DNA Miniprep Column. Discard the
flow-through liquid and place the HiBind DNA Mini Column back into the same
collection tube.
2)
Add 500 μl of HB Buffer and centrifuge
at 13,000 x g for 30 to 60 seconds at room temperature to wash the HiBind DNA
Mini Column. Discard the flow-through liquid and place the HiBind DNA Mini
Column back into the same collection tube. This step ensures that residual
protein contaminations are removed, thus ensuring high quality DNA that will be
suitable for downstream applications.
3)
Add 700 μl of DNA Wash Buffer (diluted
with absolute ethanol) and centrifuge at 13,000 x g for 30 to 60 seconds at
room temperature to wash the HiBind DNA Column. Discard the flow-through liquid
and place the HiBind DNA Mini Column back into the same collection tube.
NOTE: DNA Wash Buffer Concentrate must be
diluted with absolute ethanol before use.
10.
OPTIONAL: Repeat wash step 10 with
another 700 μl of DNA Wash Buffer (diluted with absolute ethanol).
11.
Centrifuge the empty HiBind Mini Column
at 13,000 x g for 2 min to dry the column
IMPORTANT: Do not skip this step - it is
critical for good yields
12.
Place the HiBind DNA Mini Column into a
new/clean 1.5 ml microcentrifuge tube (not supplied). Depending on desire
concentration of final product, add 30-100 μl of Elution Buffer (10 mM
Tris-HCl, pH 8.5) or sterile deionized water directly onto the center of the
column matrix. Incubate at room temperature for 1 minute. Centrifuge for at
13,000 x g for 1 min to elute DNA. An optional second elution will yield any residual
DNA, though at a lower concentration.
13.
Yield and quality of DNA: Determine the
absorbance of an appropriate dilution of the sample at 260 nm and then at
280nm.
Ⅳ. Transformation
(heat-shock method)
1.
Add DNA (Plasmid: 1ul; Ligation: all) to
each tube in which 100ul newly prepared competent cells are stored. Mix the
contents of the tubes by swirling gently. Ensure that the competent cells are
stored on the ice all the time.
2.
(Negative control: 50uL DH5αcompetent
cells+1uL sterile H2O)
3.
Store the tubs on ice for 30 minutes.
4.
Heat shock：Transfer
the tubes to a rack placed in a preheated 42℃ circulating
water bath. Store the tubes in the rack for exactly 90 seconds. Do not shake
the tubes.
5.
Rapidly transfer the tubes to an ice
bath. Allow the cells to chill for 1-2 minutes (no more than 2 minutes)
6.
Add 200ul-500ul of LB medium (sterile
and without antibiotics),Incubate the cultures for 45minutes with gentle
agitation (100-120rpm) at 37℃ to allow the bacteria
to recover and to express the antibiotic resistance marker encoded by the
plasmid.
7.
Harvest the cells by centrifugation at 2500rpm
for 2minutes.
8.
Discard some of the supernatant and
resuspend the pellets with the left 100ul of LB medium.
9.
Transfer all of the transformed
competent cells onto agar LB medium containing appropriate antibiotic. (Preheat
the plate in 37℃ incubator).
10.
Store the plates at room temperature
until the liquid has been absorbed.
11.
Invert the plates and incubate at 37℃.
Transformed colonies should appear in 12-16 hours.
Attention:
[1]
Operate gently throughout the whole
procedure.
[2]
Include all of the appropriate positive
and negative controls every time. Negative controls：Add
ddH2O instead of DNA.
Positive controls：Add
pure plasmid extraction instead of ligation.
[3]
Had better use newly made ice instead of
the ice box that has been stored at -20℃, in case of
killing cells.
[4]
Strictly control the time of heat shock
[5]
Store the tubes on ice after heat shock
no more than 2min；
[6]
The optimizing time of recovery is
45minutes. Ensure that the shaking speed should not be too fast (had better no
more than 100rpm).
[7]
The agar LB medium should be preheated
at 37℃
Ⅴ.Cleavage with two
restriction enzymes
System：40ul
|
Restriction
enzyme 1
|
0.5ul
|
|
BSA(optional)
|
4ul
|
|
Restriction
enzyme 2
|
0.5ul
|
|
Corresponding
Buffer
|
4ul
|
|
DNA
|
According to
the concentration of the plasmid to be cleaved
|
|
ddH2O
|
To meet 40ul
|
Incubate the
reaction mixtures for 2 hours at 37℃
Attention:
[1]
The efficiency of SpeI is relatively low,
therefore the amount of DNA can be decreased. The efficiency of EcoRⅠand
XbaⅠis
relatively high, therefore the enzymes can be diluted two-three fold.
[2]
The type of buffer should be selected
according to the restriction enzymes.
[3]
The amount of DNA should not exceed 4ug；
[4]
The enzymes should be stored on the ice
all the time. The buffer should be used after being thawed and blended.
[5]
Quick operation is recommended.
Ⅵ. Gel Extraction
Things
to do before starting:
• Dilute SPW Wash Buffer with absolute
ethanol
• Set heating block or water bath to
60°C
1.
Perform agarose gel/ethidium bromide
electrophoresis to fractionate DNA fragments. Any type or grade of agarose may
be used. However, it is strongly recommended that fresh TAE buffer or TBE
buffer be used as running buffer. Do not reuse running buffer as its pH will
increase and reduce yields.
2.
When adequate separation of bands has
occurred, carefully excise the DNA fragment of interest using a wide, clean,
sharp scalpel. Minimize the size of the gel slice by removing extra agarose.
3.
Determine the appropriate volume of the
gel slice by weighing it in a clean 1.5 ml microcentrifuge tube. Assuming a
density of 1g/ml of gel, the volume of gel is derived as follows: a gel slice
of mass 0.3 g will have a volume of 0.3 ml.
4.
Add an equal volume of Binding Buffer
(XP2). Incubate the mixture at 60°C for 7 min or until the gel has completely
melted. Mix by shaking or vortexing the tube in increments of 2-3 minutes.
IMPORTANT: Monitor the pH of the
Gel/Binding Buffer mixture after the gel has completely dissolved. DNA yields
will significantly decrease when the pH > 8.0. If the color of the mixture
becomes orange or red, add 5 μl of 5M sodium acetate (pH 5.2) to bring the pH
down. After this adjustment, the color of the Gel/Binding Buffer mixture should
be light yellow.
5.
Place a HiBind DNA Mini Column in a
provided 2 ml collection tube.
6.
Add the DNA/agarose solution from step 3
to the HiBind DNA Mini Column and centrifuge at 10, 000 x g for 1 min at room
temperature. Discard the flow-through liquid and place the HiBind DNA Mini
Column back into the same collection tube.
Note: For volumes greater than 700 μl,
load the column and centrifuge successively, 700 μl at a time. Each HiBind DNA
Mini Column has a total capacity of 25 μg DNA. If the expected yield is larger,
divide the sample into the appropriate number of columns.
7.
Add 300 μl of Binding Buffer (XP2) into
the HiBind DNA Mini Column. 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.
8.
Add 700 μl of SPW Wash Buffer (diluted
with absolute ethanol) to the HiBind DNA Mini Column. Centrifuge at 13,000 x g
for 1 min at room temp to wash the HiBind DNA Mini Column. Discard the flow-through
liquid and place the HiBind DNA Mini Column back into the same collection tube.
IMPORTANT: SPW Wash Buffer must be diluted
with absolute ethanol before use.
Refer to label for instructions. If
refrigerated, SPW Wash Buffer must be brought to room temperature before use.
9.
OPTIONAL: Repeat steps 7 with
another 700 μl of SPW Wash Buffer.
10.
Perform the second wash step for any
salt sensitive downstream applications
11.
Centrifuge the empty HiBind DNA Mini
Column for 2 min at maximal speed ( ≥13,000 x g ) to dry the column matrix. Do
not skip this step, it is critical for the removal of ethanol from the HiBind
DNA column.
12.
Place the HiBind DNA Mini Column into a
clean 1.5 ml microcentrifuge tube.
13.
Depending on desire concentration of the
final product, add 30-50 μl Elution Buffer (10 mM Tris-HCl, pH 8.5) or water
directly onto the column matrix and incubate at room temperature for 2 minutes.
Centrifuge for 1 min at maximum speed (≥13,000 x g) to elute DNA.
14.
This represents approximately 70% of
bound DNA. An optional second elution will yield any residual DNA, though at a
lower concentration.
NOTE: The efficiency of eluting DNA from
the HiBind DNA Mini Column is dependent of pH. If eluting DNA with deionized
water, make sure that the pH is around 8.5.
15.
Yield and quality of DNA: Determine the
absorbance of an appropriate dilution of the sample at 260 nm and then at
280nm.
Ⅶ. Identification
of DNA by Electrophoresis
Marker:
λDNA/HindⅢ+EcoRⅠ
DL 2000
Ⅷ. Ligation
System：30ul
|
T4 Ligase
|
1ul
|
|
10XBuffer
|
3ul
|
|
DNA fragment
|
According to
the concentration of the DNA fragment
|
|
Vector
|
According to
the concentration of the vector
|
|
ddH2O
|
To meet 30ul
|
Incubate the
reaction mixtures at 37℃
for 2-3 hours.
Attention：
[1]
The ratio of DNA fragments to vector
should be 3-5:1
[2]
The ligation time should not be too
long.
[3] If
there are several DNA fragments to be ligated to the same vector, the sequence
should be optimized according to actual conditions.
Ⅸ. PCR of the colony
System：20ul
Taq
0.5ul
Primer
1
0.6ul
Primer
2
0.6ul
dNTP
0.8ul
Template
1ul
DMSO
1.2ul
10 X
Buffer
2ul
ddH2O
13.3ul
Reaction
condition：
94℃
5min
94℃
30sec
55-68℃
30sec
72℃
1min
72℃
7min
28cycles
Attention：
[1] Pick
ten single colonies for each cloning.
[2] The
PCR mix should be a little more than needed, in case of the loss during
operation.
[3] DMSO
should be added into each PCR tube separately.
[4] The
PCR mix should be blended.
Ⅹ.Identification
of DNA by Sequencing
Send the
plasmids or the cell culture to BGI (A sequencing company in China) for
sequencing.
XI.Others
1、LB
medium components
|
Volume
|
100ml liquid medium
|
100ml solid medium
|
|
Trptone
|
1g
|
1g
|
|
Yeast Extraction Powder
|
0.5g
|
0.5g
|
|
NaCl
|
1g
|
1g
|
|
Agar
|
|
1.5g
|
2、Amp
mother solution
Preparation：Add
100mg ampicillin into 1mlddH2O
Usage：Add
50ulmother solution into 100ml medium
3、Kana
mother solution
Preparation：Add
50mg kanamycin into 1mlddH2O
Usage：Add
100ulmother solution into 100ml medium
4、Chl
mother solution
Preparation：Add
50mg kanamycin into 1mlddH2O
Usage：Add
100ulmother solution into 100ml medium
"
