Biology Reference
In-Depth Information
3.3. Cloning of PCR Products
1.
Prior to cloning the PCR product, remove excess primers using a spin column
(QIAquick PCR Purification Kit) and elute the purified PCR product in 50
µ
L of
Tris-HCl pH 7.5.
2.
Set up a ligation reaction using 5
µ
L of purified PCR product, 1
µ
L of 10X ligase
buffer, 2
µ
L of TA vector DNA and 2
µ
L of sterile dH
2
O. Incubate the reaction
overnight at 4
°
C.
3.
Thaw one vial of competent cells (50
µ
L) for each ligation and place on ice. Add
5
-mercaptoethanol. Allow the transforma-
tion to proceed on ice for 30 min. Heat shock the cells at 42
µ
L of ligation reaction and 1
µ
L of
β
°
C for 30 s and place
back on ice for 2 min. Add 250
µ
L of SOC medium and incubate at 37
°
C for 1 h
in a shaking incubator.
4.
Spread transformation onto LB agar plates containing 50 mg/mL Carbenicillin
and X-Gal. Incubate inverted at 37
°
C for 16-18 h.
5.
Using a sterile pipet tip pick 10 recombinant (white) colonies and with each one
inoculate 5 mL LB broth containing 50
µ
g/mL Carbenicillin. Grow overnight at
37
°
C in a shaking incubator.
6.
Isolate plasmid DNA from 1.5 mL of overnight culture using a QIAprep Spin
Miniprep Kit. Elute purified plasmid DNA in 100
µ
L of 10 m
M
Tris-HCl pH 7.5.
7.
Digest 10 mL of purified plasmid DNA with
Eco RI
(or other suitable restriction
endonuclease) to confirm presence of DNA insert.
3.4. DNA Sequence Analysis of Cloned PCR Products
1.
Quantify the amount of plasmid DNA using an OD
260
spectrophotometer mea-
surement and dilute to 50 ng/
µ
L with 10 m
M
Tris-HCl pH 7.5.
2.
Set up following sequencing reactions:
Plasmid DNA (50 ng/
µ
L)
8
µ
L (400 ng)
Terminator ready mix
2
µ
L
Reaction buffer
6
µ
L
(450 m
M
Tris-HCl pH 8.0, 10 m
M
MgCl
2
)
Primer (0.8 pmols/mL)
L (3.2 pmols)
(M13 Reverse primer or M13 Forward (-20) primer—
see
Note 5
)
4
µ
3.
Overlay the reaction with light mineral oil and use the following 25 cycle
sequencing parameters:
a. 96
°
C for 30 s (template denaturation).
b. 50
°
C for 30 s (primer annealing).
c. 60
°
C for 4 min (extension).
4.
Remove sequencing reaction products from under the mineral oil and place in a
clean microcentrifuge tube. Add 2
µ
L of 3M NaOAc pH 4.6 and 50
µ
L of ethanol
(99%), vortex and incubate on ice for 10 min.
5.
Spin samples at 13,600 xg in a microcentrifuge for 15 min then carefully remove
the supernatant with a pipet.
Search WWH ::
Custom Search