Team:Tsinghua/Notebook

From 2008.igem.org

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2.2 Program:
2.2 Program:
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20. Extract plasmid and send sequencing
20. Extract plasmid and send sequencing
End^^67
End^^67
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part number plasmid cell strain concentration
 
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BBa_K108000 pSB1AC3 TOP10 100ng/uL
 
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BBa_K108001 pSB1AC3 TOP10
 
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BBa_K108002 pSB1AC3 TOP10
 

Revision as of 11:15, 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

1. 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


2. Fusion PCR

2.1 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.

2.2 Program:

Fusion PCR Program
Step Condition Time Remark
1 95℃ 5min 5
2 95℃ 30-50sec 0.3
3 95℃ 10mM each 1
4 95℃ 10uM 1
5 95℃ 10um 1
6 changeable 0.5 1
7 0.2M 0.5 1
8 40.5 81

95℃ 5min

95℃ 30-50sec

{Tm(fu)+[(-2)~5]}℃ 40-80sec

72℃ DNA length/kb/min

RETURN TO LINE 2 FOR 10-15 cycles

72℃ 5min

Add amplification Primers

95℃ 2-5min

continue common program for 25-30 cycles


3. Restriction Digestion

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).


4. 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

BioBrickTM Parts Making Protocol 1. get desired sequences through NCBI or other sources and check for restriction sites For no (Xba1 or Spe1) Goto STEP2 Else Goto STEP9 2. Design primers with half-prefix (Xba1) and half-suffix (Spe1) 3. PCR from according genome/plasmid 4. Purify PCR product using Gel Extraction Kit (Transgen) 5. Digest with Xba1+Spe1 6. Ligation with pSB1AC3, which was digest with Xba1+Spe1 and treated with CIAP 7. Transform to TOP10 cells 8. Identify clones with colony PCR Goto STEP20 9. Design primers with full-prefix and full-suffix 10. PCR from according genome/plasmid 11. Purify PCR product using Gel Extraction Kit (Transgen) For EcoR1 Goto STEP12 Else Goto STEP14 12. Digest with Xba1+Pst1 13. Ligation with pSB1AC3, which was digest with Xba1+Pst1 and treated with CIAP Goto STEP16 14. Digest with EcoR1+Spe1 15. Ligation with pSB1AC3, which was digest with EcoR1+Spe1 and treated with CIAP 16. Transform to TOP10 cells 17. Identify clones with colony PCR 18. Extract plasmid and site-directed mutate by fusion PCR 19. Transform to TOP10 cells 20. Extract plasmid and send sequencing End^^67



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