Team:HokkaidoU Japan/Notebook/September3

From 2010.igem.org

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=1-3AのPCR=
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=PCR of 1-3A=
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1-3AはpSB1C3に載ったRFP reporterで，トラフォメに成功している<br>
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ベクターとして配布されたpSB1C3が悪い可能性を見るため，このパーツのpSB1C3を増幅して使用する
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Transformation of 1-3A part which is originaly on pSB1C3 was succesful<br>
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Thinking that linearized vector might gone bad we PCRed pSB1C3 from 1-3A
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{|style="text-align:center;" class="protocol"
!Reagent
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* extensionは120 sec
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* Extension was for120 sec
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* YM-10でClean Upしたら43 uLになった
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* Purified via Microcon YM-10
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* Final volume was 43 uL

Revision as of 17:34, 27 September 2010

Notebook

Resultsof yesterdays trnsformation

  • pSB1C3 uterly failed to produce colonies
  • pUC119 produced 20 colonies
  • colonies that should been red because of RFP insert wasn`t, so there is posibility that insert wasn`t there

Colony PCR on yesterdays E.coli

  • Colony PCR was done acordig to protocol
  • This day we did 20 samples

Electrophoresis

Concentration check of parts used for transformation yesterday

Electrophoresis of parts before ligation

Confirmed that DNA solution concentration of parts for Ligation was as anticipated (good?)

Lane DNA
1
2 Lambda/Hind III, EcoR I
3
4 RFP
5 pUC119
6 pSB1C3

PCR of 1-3A

Transformation of 1-3A part which is originaly on pSB1C3 was succesful
Thinking that linearized vector might gone bad we PCRed pSB1C3 from 1-3A

Reagent Amount
1-3A 1
DW 33
10x Buffer 5
2 M 4dNTPs 5
25 mM MgSO4 3
Suffix-F 1
Prefix-R 1
KOD 1
Total 50 uL
  • Extension was for120 sec
  • Purified via Microcon YM-10
  • Final volume was 43 uL
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