Team:KAIT Japan/Protocol

From 2012.igem.org

(Difference between revisions)
Line 54: Line 54:
Editing now.
Editing now.
-
----
+
=Colony PCR=
-
===Colony PCR===
+
Reagent
Reagent
:*TaKaRa Ex Taq(5units/μL) 0.5μL
:*TaKaRa Ex Taq(5units/μL) 0.5μL
Line 74: Line 73:
#*gradient:57-62°C(+0.1c)
#*gradient:57-62°C(+0.1c)
----
----
-
===Ligation===
+
=Ligation=
Reagent
Reagent
:*sterilize water 2μL
:*sterilize water 2μL
Line 84: Line 83:
#Storage Overnight(-4°C)
#Storage Overnight(-4°C)
----
----
-
===DNA extraction and purification of ''P.aeruginosa''===
+
=DNA extraction and purification of ''P.aeruginosa''=
#Centrifuge culture medium(6,000rpm,5min,4°C)
#Centrifuge culture medium(6,000rpm,5min,4°C)
#Remove supernatant,Add saline[0.85%](1.5mL)
#Remove supernatant,Add saline[0.85%](1.5mL)
Line 120: Line 119:
#*Melt DNA in buffer
#*Melt DNA in buffer
----
----
-
===Transformation===
+
=Transformation=
#Put competent cells on ice(10-15min)
#Put competent cells on ice(10-15min)
#Add Ligation reaction solution(10μL) and tapping
#Add Ligation reaction solution(10μL) and tapping
Line 130: Line 129:
#Cultivation(overnight)
#Cultivation(overnight)
----
----
-
===Confirmed of electrophoresis by PCR product and Ligation of the TA vector===
+
=Confirmed of electrophoresis by PCR product and Ligation of the TA vector=
#Electrophoresis
#Electrophoresis
#*Gel concentration:1.2%,Migration time:30min
#*Gel concentration:1.2%,Migration time:30min
Line 144: Line 143:
#Storage(-20°C)
#Storage(-20°C)
----
----
-
===The purified DNA===
+
=The purified DNA=
#Electrophoresis
#Electrophoresis
#*Marker:dye 1μL,DNA molecule 2μL,TE buffer 3μL
#*Marker:dye 1μL,DNA molecule 2μL,TE buffer 3μL
Line 152: Line 151:
#Storage
#Storage
----
----
-
===PCR Product===
+
=PCR Product=
#Electrophoresis
#Electrophoresis
#*The gel check and cut
#*The gel check and cut
Line 161: Line 160:
#Storage
#Storage
----
----
-
===Miniprep===
+
=Miniprep=
#Add culture medium 1mL in a microtube.
#Add culture medium 1mL in a microtube.
#Centrifuge(1min,4°C,12,000rpm).
#Centrifuge(1min,4°C,12,000rpm).

Revision as of 08:18, 31 August 2012

KAIT Japan2012 logo.png
KAIT Japan2012 Sub.png
Kaitjapan iGEM official.logo.png


Kaitjapan.home.png

Home

Kaitjapan project.png

Project

Kaitjapan parts.png

Parts

Kaitjapan protocol.png

Protocol

Kaitjapan notebook.png

Notebook

Kaitjapan results.png

Results

Kaitjapan safety.png

Safety

Kaitjapan human practice.png

Human Practice

Kaitjapan team.png

Team

Editing now.

Contents

Colony PCR

Reagent

  • TaKaRa Ex Taq(5units/μL) 0.5μL
  • 10×Ex Taq buffer 10μL
  • dNTP Mixture(2.5Meach) 8μL
  • Primer F(10μM) 4μL
  • Primer R(10μM) 4μL
  • Template(E.coli DH5α)
  • sterilized water(73.5μL)

Conditions of the thermal cycler

  1. 95°C(5min)
  2. 94°C(30sec)
  3. 61°C(30sec)
  4. 71°C(40sec)
  5. 72°C(1min)
  6. 4°C(Save)
    • 2-4:30cycle
    • gradient:57-62°C(+0.1c)

Ligation

Reagent

  • sterilize water 2μL
  • PCR product 2μL
  • vector DNA 1μL
  • Ligation Mighty Mix 5μL

Method

  1. Incubation(1h,16°C)
  2. Storage Overnight(-4°C)

DNA extraction and purification of P.aeruginosa

  1. Centrifuge culture medium(6,000rpm,5min,4°C)
  2. Remove supernatant,Add saline[0.85%](1.5mL)
  3. Centrifuge(6,000rpm,5min,4°C)
  4. Add 5mMEDTA 1mL
  5. Add 10%SDS 100μL
  6. Add proteinase K 50methodL
  7. Vortex
  8. Incubation(30min,55°C)
  9. Add phenol mixture(TE saturated phenol:chloroform:isoamyl alcohol=25:24:1)
  10. Shake vigorously(1min)
    • At this time,It became muddy white in color.
  11. Centrifuge(16,000rpm,10min,4°C)
  12. Pick up supernatant,remove new microtube
  13. Repeat step 7-11
  14. Add 3M-sodium acetate 40μL,chilled isopropanol 400μL
  15. Vortex
  16. Wind the DNA by a thin glass rod.
  17. Rinse chilled 70%-ethanol(500μL)
  18. Pick up DNA,air dry
  19. Add TE buffer 500μL
  20. Add RNase A 50μL
  21. Incubation(20min,37°C)
  22. Add proteinase K 50μL
  23. Incubation(1h,37°C)
  24. Add phenol mixture
  25. Vortex(1min)
  26. Centrifuge(16,000rpm,10min,4°C)
  27. Pick up supernatant,remove new microtube
  28. Add 3M-sodium acetate 40μL,chilled isopropanol 400μL
  29. Wind the DNA by a thin glass rod.
  30. Rinse chilled 70%-ethanol(500μL,about 30s)
  31. Pick up DNA,air dry
  32. Add TE buffer 200μL
    • Melt DNA in buffer

Transformation

  1. Put competent cells on ice(10-15min)
  2. Add Ligation reaction solution(10μL) and tapping
  3. On the ice(30min)[Transformation]
  4. Add LB medium(0.7mL)
  5. Incubate(60min,37°C)
  6. Add X-gal(40μL) and ampicillin(10μL)[200μg/mL] on LB agar medium(IPTG)
  7. Add one incubated(100μL)
  8. Cultivation(overnight)

Confirmed of electrophoresis by PCR product and Ligation of the TA vector

  1. Electrophoresis
    • Gel concentration:1.2%,Migration time:30min
    • Marker:Flash Gel 5μL
    • Sample:dye 1μL,sample 5μL
  2. Check and Colony PCR
  3. Add to TA vector
    • PCR product 2μL
    • pMD20-Tvector 1μL
    • D2W 2μL
    • Ligation Mighty Mix 5μL
  4. Heat insulation(16°C,30min)
  5. Storage(-20°C)

The purified DNA

  1. Electrophoresis
    • Marker:dye 1μL,DNA molecule 2μL,TE buffer 3μL
    • Sample:dye 1μL,sample 5μL
    • Gel concentration:1.2%,Migration time:30min
→Reflection:Band was less.
  1. Storage

PCR Product

  1. Electrophoresis
    • The gel check and cut
  2. DNA purification
  3. Confirmation of electrophoresis
  4. PCR
    • 50cycle
  5. Storage

Miniprep

  1. Add culture medium 1mL in a microtube.
  2. Centrifuge(1min,4°C,12,000rpm).
  3. Remove the supernatant.
  4. Repeat 1-3.
  5. Add SolI 100μL and Vortex.
  6. Centrifuge(1min,4°C,12,000rpm).
  7. Add SolII 200μL and invert.
  8. ice-cold 3min.
  9. Add SolIII 150μL and invert.
  10. ice-cold 5min.
  11. Centrifuge(5min,4°C,12,000rpm).