Team:HUST-China/wetlab/protocols

「Protocols」

• Cloning methods
• Reaction System
  of Colony PCR
• SDS-PAGE

Sensing part

A.The PCR Reaction System

Components (50μL)	Volume(μL)
5×phusion HS Buffer	10
dNTPs(2.5mM)	5
Primer-F(10μM)	2.5
Primer-R(10μM)	2.5
DMSO	1.5
phusion	0.5
ddH2O	27.5
Template	0.5

B.The double enzyme digestion system (Q.cut)

Components (30μl)	Volume(μl)
10 x FD buffer	3
dNTPs(2.5mM)	5
Enzyme I	1
Enzyme II	1
ddH2O	1.5
Conditions	37℃ 1.5~2h

C.Ligation system

Components (10μl)	Volume(μl)
T4 ligase	1
T4 ligase buffer	1
Linearized Vector	1
Insert Gene	7
Conditions	16°C 1h

D.The Bridging PCR protocols

Bridge PCR: take normal protocol with following revision: PCR 2 bridge DNA sections with upper/midup or midlow/lower primers from model, midup and midlow primer should be designed overlap with at least 22 bp, PCR result DNA sections can be used together as models for 2nd PCR with upper/lower primers.

E.Transformation into E. coli DH5α

The ligation product was transformed into E.coli DH5α strain.

If the vector was pET28a, the strain was grown in LB plate medium containing 10μg/ml kanamycin at 37°C.

If the vector was pBAD30, the strain was grown in LB plate medium containing 100μg/ml ampicillin at 37°C.

If the vector was pSB1C3, the strain was grown in LB plate medium containing 25μg/ml chloramphenicol at 37°C.

Reaction System of Colony PCR

Components (20μl)	Volume(μl)
Es Taq Mix(2×)	10
Primer-F(10uM)	0.55
Primer-R(10uM)	0.55
ddH2O	8.9

Construction of the whole circuit

The protocol of SDS-PAGE:

a.Centrifuge 1ml culture medium/bacteria liquid at 12000rmp for 2 min to separate the supernatant and cells.

b.Add 100μl DNase/RNase-Free Water to the precipitation, then add in 100μl 2×loading buffer.

c.Place tubes in 100℃ heat block for 10 min.

d.Centrifuge at 12000rmp for 2 min.

e.Do SDS-PAGE

Acknowledgments: