Biology Reference
In-Depth Information
2.2 Cloning
1. TE buffer: 10 mM Tris-HCl (pH 8), 1 mM EDTA.
2. Enzymes:
-restriction enzymes:
Eco
RI,
Age
I.
-ligase, e.g., T4 DNA Ligase, 1 U/
L, Invitrogen.
-proof-reading polymerase, e.g., Phusion
®
High-Fidelity
DNA Polymerase, NEB.
-Taq polymerase.
3. Gel extraction kit, e.g., peqGOLD Gel Extraction Kit (C-Line),
Peqlab.
4. dNTPs (10 mM each) in water.
5. TAE buffer: 1 mM EDTA, 40 mM Tris (pH 8.3), 20 mM
acetic acid. TAE buffer is commonly prepared as a 50× stock
solution: dissolve 242 g Tris base in water, add 57.1 mL glacial
acetic acid and 3.3 g of disodium ethylenediaminetetraacetate
×2H
2
O, and bring the fi nal volume up to 1 l. This stock solu-
tion can be diluted 50:1 with water to make a 1× working
solution.
6. SB (sodium boric acid) buffer: 5 mM disodium borate decahy-
drate. A 25× stock solution is prepared by dissolving 95.34 g
Na
2
B
4
O
7
·10 H
2
O in 2 L H
2
O.
7. Agarose gels (1-1.5 %) in 1× TAE buffer: Weigh 1-1.5 g of
agarose in a 250 mL Erlenmeyer fl ask (
see
Note 1
) and boil it
in 100 mL 1× TAE buffer. Add 1
μ
L ethidium bromide
(10 mg/mL; Fluka) just before the solution is lukewarm, mix
it cautiously and pour it into a gel casting tray (
see
Note 2
).
As running buffer use 1× TAE buffer.
8. Agarose gels (2 %) in 1× SB buffer: Boil 2 g of agarose in
100 mL SB buffer as in previous step.
9. 5× TAE loading buffer: Dissolve 45 g Ficoll 70, ~20 mg bro-
mophenol blue and 2.25 g
N
-lauroylsarcosin in 50 mL H
2
O.
Add 3.6 mL glycerol and 9 mL 50× TAE buffer. Adjust the
volume to 90 mL with H
2
O.
10. 5× SB loading buffer: Dissolve 45 g Ficoll 70, ~20 mg xylene
cyanol, and 2.25 g
N
-lauroylsarcosin in 50 mL H
2
O. Add
3.6 mL glycerol and 18 mL 25× SB buffer. Adjust the volume
to 90 mL with H
2
O.
11. Antibiotics (bacteria): Dissolve ampicillin or carbenicillin in
water at 100 mg/mL and store at −20 °C in aliquots.
12. LB-Medium: Suspend 20 g LB-Broth in 1 L of water and
autoclave for 15 min at 121 °C. Storage at 4 °C or room tem-
perature (
see
Note 3
).
13. LB-agar: Suspend 705 g LB-Agar in 2 L water, aliquot
400 mL each with a stir bar in 0.5 L fl asks and autoclave for
μ
