Team:HokkaidoU Japan/Notebook/September23

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*Electroporetion of BAC plasmid into DH5α MG1655
*Electroporetion of BAC plasmid into DH5α MG1655
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== Bac Vecter Purification ==
+
= Bac Vecter Purification =
Used Qiagen miniprep kit, qiaprep for miniprep
Used Qiagen miniprep kit, qiaprep for miniprep
#Transfered 1.3 mL of BAC plasmid solution incubated overnight into 1.5 mL tube
#Transfered 1.3 mL of BAC plasmid solution incubated overnight into 1.5 mL tube
-
#Centrifuged at 4C, 15000rpm for 1min
+
#Centrifuged at 4C, 15000 rpm for 1 min
#Discarded the supernatant and added remaining solution.
#Discarded the supernatant and added remaining solution.
-
#Centrifuged at 4C, 15000rpm for 1min
+
#Centrifuged at 4C, 15000 rpm for 1 min
#Discarded the supernatant
#Discarded the supernatant
#Suspended on 250 uL of Buffer P1
#Suspended on 250 uL of Buffer P1
#Added 250 ul Buffer P2 inverted few times to mix, solution turned green
#Added 250 ul Buffer P2 inverted few times to mix, solution turned green
#Added 350 ul Buffer N3 mixed by inversion, precipitation apeared
#Added 350 ul Buffer N3 mixed by inversion, precipitation apeared
-
#Centrifuged at 4C, 13000rpm for 10min
+
#Centrifuged at 4C, 13000 rpm for 10 min
#Transfered the supernatant to filtration column  
#Transfered the supernatant to filtration column  
-
#Centrifuged at 4C, 13000rpm for 1min
+
#Centrifuged at 4C, 13000 rpm for 1 min
#Discarded the flow-through
#Discarded the flow-through
#added 500 uL of Buffer PB  to filtration column  
#added 500 uL of Buffer PB  to filtration column  
-
#Centrifuged at 4C, 13000rpm for 1min
+
#Centrifuged at 4C, 13000 rpm for 1 min
#Discarded the flow-through centrifuged for 1min to remove remaining buffer
#Discarded the flow-through centrifuged for 1min to remove remaining buffer
#Transfered filtration column to a new 1.5 ml tube
#Transfered filtration column to a new 1.5 ml tube
#Resuspended on 50 ul of TE and incubated at RT for 1min
#Resuspended on 50 ul of TE and incubated at RT for 1min
-
#Centrifuged at 4C, 13000rpm for 1min
+
#Centrifuged at 4C, 13000 rpm for 1 min
-
#Stored at -20
+
#Stored at -20C
-
== AraC+RBS+pSB1A3のコロニーPCR ==
+
= Colony PCR of AraC+RBS+pSB1A3 =
-
#コロニーを無作為に16個選び、番号を振った。
+
*Selected 16 colonies at random, and PCRed them.  
-
#0.5 mLチューブを16本用意し、それぞれにDWを10 uLずつ分注した。
+
*PCR mix used is shown in the table bellow
-
#チューブに番号を振り、対応するコロニーを懸濁した。
+
-
#コロニーPCR溶液を以下の組成に従って調整した。
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{|style="text-align:center; margin-left:100px;" class="protocol"
{|style="text-align:center; margin-left:100px;" class="protocol"
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|-
|-
|Taq Master Mix
|Taq Master Mix
-
|90 uL
+
|80 uL
|-
|-
|Ex-F
|Ex-F
-
|1.8 uL
+
|1.6 uL
|-
|-
|Ps-R
|Ps-R
-
|1.8 uL
+
|1.6 uL
|-
|-
|style="border-top:1px solid #996;"|'''Total'''
|style="border-top:1px solid #996;"|'''Total'''
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|style="border-top:1px solid #996;"|'''93.6 uL'''
+
|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

  • Amplifiable BAC plasmid (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.