Team:HokkaidoU Japan/Notebook/September23

From 2010.igem.org

(Difference between revisions)
 
(10 intermediate revisions not shown)
Line 1: Line 1:
{{Template:HokkaidoU_Japan}}
{{Template:HokkaidoU_Japan}}
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September22|September 22]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September24|September 24]]</div></div>
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September22|September 22]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September24|September 24]]</div></div>
 +
 +
*Amplifiable BAC plasmid ([http://partsregistry.org/Part:BBa_J61031 BBa_J61031]) purification
 +
*Colony PCR of AraC+RBS+pSB1A3
 +
*Electroporetion of BAC plasmid into DH5α MG1655
 +
 +
= Bac Vecter Purification =
 +
 +
Used Qiagen miniprep kit, qiaprep for miniprep
 +
 +
#Transfered 1.3 mL of BAC plasmid solution incubated overnight into 1.5 mL tube
 +
#Centrifuged at 4C, 15000 rpm for 1 min
 +
#Discarded the supernatant and added remaining solution.
 +
#Centrifuged at 4C, 15000 rpm for 1 min
 +
#Discarded the supernatant
 +
#Suspended on 250 uL of Buffer P1
 +
#Added 250 ul Buffer P2 inverted few times to mix, solution turned green
 +
#Added 350 ul Buffer N3 mixed by inversion, precipitation apeared
 +
#Centrifuged at 4C, 13000 rpm for 10 min
 +
#Transfered the supernatant to filtration column
 +
#Centrifuged at 4C, 13000 rpm for 1 min
 +
#Discarded the flow-through
 +
#added 500 uL of Buffer PB  to filtration column
 +
#Centrifuged at 4C, 13000 rpm for 1 min
 +
#Discarded the flow-through centrifuged for 1min to remove remaining buffer
 +
#Transfered filtration column to a new 1.5 ml tube
 +
#Resuspended on 50 ul of TE and incubated at RT for 1min
 +
#Centrifuged at 4C, 13000 rpm for 1 min
 +
#Stored at -20C
 +
 +
 +
= Colony PCR of AraC+RBS+pSB1A3 =
 +
*Selected 16 colonies at random, and PCRed them.
 +
*PCR mix used is shown in the table bellow
 +
 +
{|style="text-align:center; margin-left:100px;" class="protocol"
 +
!Reagent
 +
!Amount
 +
|-
 +
|Taq Master Mix
 +
|80 uL
 +
|-
 +
|Ex-F
 +
|1.6 uL
 +
|-
 +
|Ps-R
 +
|1.6 uL
 +
|-
 +
|style="border-top:1px solid #996;"|'''Total'''
 +
|style="border-top:1px solid #996;"|'''83.2 uL'''
 +
|}
 +
 +
[[Image:9.23 coloP.JPG‎|200px|right|thumb|Result of colony PCR]]
 +
 +
*electrophoresed the samples
 +
 +
1 lane -> Marker λ/HindIII EcoRI 5 uL
 +
 +
2~8 lane -> colony No.1~7
 +
 +
9 lane -> Marker λ/HindIII EcoRI 5 uL
 +
 +
10~16 lane -> colony No.8~14
 +
 +
 +
Colony No.15,16 didn't electrophorese.
 +
 +
 +
*Colony No.1 will have the plasmid,so we put the sample into 2 mL LB added 2 uL Ampicillin(100 ug/uL).Incubated it in a shaker at 37C, 180 rpm.

Latest revision as of 18:02, 27 October 2010

Notebook
  • Amplifiable BAC plasmid ([http://partsregistry.org/Part:BBa_J61031 BBa_J61031]) purification
  • Colony PCR of AraC+RBS+pSB1A3
  • Electroporetion of BAC plasmid into DH5α MG1655

Bac Vecter Purification

Used Qiagen miniprep kit, qiaprep for miniprep

  1. Transfered 1.3 mL of BAC plasmid solution incubated overnight into 1.5 mL tube
  2. Centrifuged at 4C, 15000 rpm for 1 min
  3. Discarded the supernatant and added remaining solution.
  4. Centrifuged at 4C, 15000 rpm for 1 min
  5. Discarded the supernatant
  6. Suspended on 250 uL of Buffer P1
  7. Added 250 ul Buffer P2 inverted few times to mix, solution turned green
  8. Added 350 ul Buffer N3 mixed by inversion, precipitation apeared
  9. Centrifuged at 4C, 13000 rpm for 10 min
  10. Transfered the supernatant to filtration column
  11. Centrifuged at 4C, 13000 rpm for 1 min
  12. Discarded the flow-through
  13. added 500 uL of Buffer PB to filtration column
  14. Centrifuged at 4C, 13000 rpm for 1 min
  15. Discarded the flow-through centrifuged for 1min to remove remaining buffer
  16. Transfered filtration column to a new 1.5 ml tube
  17. Resuspended on 50 ul of TE and incubated at RT for 1min
  18. Centrifuged at 4C, 13000 rpm for 1 min
  19. Stored at -20C


Colony PCR of AraC+RBS+pSB1A3

  • Selected 16 colonies at random, and PCRed them.
  • PCR mix used is shown in the table bellow
Reagent Amount
Taq Master Mix 80 uL
Ex-F 1.6 uL
Ps-R 1.6 uL
Total 83.2 uL
Result of colony PCR
  • electrophoresed the samples

1 lane -> Marker λ/HindIII EcoRI 5 uL

2~8 lane -> colony No.1~7

9 lane -> Marker λ/HindIII EcoRI 5 uL

10~16 lane -> colony No.8~14


Colony No.15,16 didn't electrophorese.


  • Colony No.1 will have the plasmid,so we put the sample into 2 mL LB added 2 uL Ampicillin(100 ug/uL).Incubated it in a shaker at 37C, 180 rpm.
Retrieved from "http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September23"