Team:Tsinghua/Notebook

From 2008.igem.org

(Difference between revisions)
m
m
Line 12: Line 12:
'''Basic Wet-lab protocols'''
'''Basic Wet-lab protocols'''
-
=PCR=
+
PCR
Fusion PCR
Fusion PCR
Restriction cut
Restriction cut
Line 68: Line 68:
|}
|}
-
=''Fusion PCR''=
+
 
 +
''Fusion PCR''
(1) System
(1) System
 +
The basic system is similar to common PCR. There are some notes to raise the fusion efficiency.
The basic system is similar to common PCR. There are some notes to raise the fusion efficiency.
 +
a. Complementary region length: 15-20bp
a. Complementary region length: 15-20bp
 +
b. Raise the annealing temperature in the fusion step.
b. Raise the annealing temperature in the fusion step.
 +
(2) Program:
(2) Program:
• Program:
• Program:
 +
• 95’: 5min
• 95’: 5min
 +
•     95'  : 30-50sec
•     95'  : 30-50sec
 +
•     Tm(fu)+ (-2)~5: 40-80sec
•     Tm(fu)+ (-2)~5: 40-80sec
 +
•     72' : the longer/1kb/min
•     72' : the longer/1kb/min
10-15 cycles  
10-15 cycles  

Revision as of 10:43, 29 October 2008

HOME Team Project 1 Project 2 Parts Modelling Notebook Doodle Board

Basic Wet-lab protocols

PCR Fusion PCR Restriction cut Ligation Transformation

PCR

PCR by Pyrobest DNA polymerase
Reagent Concentration/Activity 50ul 100ul
10xPyrobest bufferII 10x 5 10
Pyrobest 0.3 0.5
dNTPmix 10mM each 1 2
Primer 1 10uM 1 2
Primer 2 10um 1 2
Template DNA changeable 0.5 1
MgCl2(Deletable) 0.2M 0.5 1
ddH2O 40.5 81


Fusion PCR (1) System

The basic system is similar to common PCR. There are some notes to raise the fusion efficiency.

a. Complementary region length: 15-20bp

b. Raise the annealing temperature in the fusion step.

(2) Program:

• Program:

• 95’: 5min

• 95'  : 30-50sec

• Tm(fu)+ (-2)~5: 40-80sec

• 72' : the longer/1kb/min 10-15 cycles • 72' 5min • Add amplification Primers • 95' 2-5min • Go on under common program for 25-30 cycles


Restriction cut

Reagent Concentration/Activity Volume(50ul system)
Restriction cut buffer 10x 5ul
Enzyme 1 1ul
Enzyme 2 1ul

Add DNA and distilled water to 50ul.Incubate at 37℃, 1.5 hrs or longer(Enzymes from Takara Co., Ltd or NEB).


Ligation

Reagent Volume(10ul system)
Solution I 5ul
DNA fragment 3.5ul(changeable)
Vector 1.5ul(changeable)

Incubate at 16-18℃,1hr or longer(Ligation kit from Takara.,Ltd).

Notes: Advanced protocol for parts extraction




  • Click on any day below to see what wet-lab procedures were conducted.