Team:HokkaidoU Japan/Notebook/August13

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{{Template:HokkaidoU_Japan}}<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/August12|August 12]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/August16|August 16]]</div></div>
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=PCR of parts which didn't amplify well via mini prep=
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==Composition of Reaction Solution==
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{|class="protocol"
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|-
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!Reagent
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!Amount
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|-
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|style="text-align:right;"| Autoclaved DW :
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|style="text-align:left;"| 33 uL
 +
|-
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|style="text-align:right;"| 10x PCR buffer :
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|style="text-align:left;"| 5 uL
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|-
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|style="text-align:right;"| 2 mM dNTPs :
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|style="text-align:left;"| 5 uL
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|-
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|style="text-align:right;"| 25 mM MgSO<sub>4</sub> :
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|style="text-align:left;"| 3 uL
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|-
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|style="text-align:right;"| EX-F primer :
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|style="text-align:left;"| 1 uL
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|-
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|style="text-align:right;"| PS-R primer :
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|style="text-align:left;"| 1 uL
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|-
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|style="text-align:right;"| KOD plus Neo :
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|style="text-align:left;"| 1 uL
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|-
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|style="text-align:right;"| Template ([[Team:HokkaidoU_Japan/Parts#BioBricks|1-18F]]) :
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|style="text-align:left;"| 1 uL
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|-
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|style="text-align:right; border-top:1px solid #996;"|'''Total''' :
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|style="text-align:left; border-top:1px solid #996;"| 50 uL
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|}
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==Removal of Primers==
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# Added 150 uL of TE to 50 uL of PCR product, each
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# Transfered into Microcon YM-10 filter and cetrifuged for 20 min at 14,000 G
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# Much of the solution remained so centrifuged for aditional 10 min
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# Measured the amount left
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## It was 140 uL so centrifuged again for 10min
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# And again
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# Finally DNA solution was reduced to 19 uL so we added 31 uL to make final volume of 50 uL
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==Electrophoresis==
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[[Image:HokkaidoU Japan 20100813a.jpg‎|200px|right|thumb|PCR for DNA confirmation]]
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# Added 0.4 uL of 6x Sample Buffer to 1 uL of DNA solution and electrophoresed it.
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# At the same time add added 2 uL of 6x Sample Buffer to 10 uL of flow trough and centrifuged
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* Used marker [https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png pUC119/''Hin''f]
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* It was confirmed that DNA was amplified by PCR
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** Between marker bands of 543 bp and 1330 bp PCR product band of 700 bp was visible
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* Due to negligence (electrophoresis for 45 min), flow through with primers flowed through and exited gel
 +
<div style="clear:both">

Latest revision as of 07:26, 27 October 2010

Notebook

PCR of parts which didn't amplify well via mini prep

Composition of Reaction Solution

Reagent Amount
Autoclaved DW : 33 uL
10x PCR buffer : 5 uL
2 mM dNTPs : 5 uL
25 mM MgSO4 : 3 uL
EX-F primer : 1 uL
PS-R primer : 1 uL
KOD plus Neo : 1 uL
Template (1-18F) : 1 uL
Total : 50 uL

Removal of Primers

  1. Added 150 uL of TE to 50 uL of PCR product, each
  2. Transfered into Microcon YM-10 filter and cetrifuged for 20 min at 14,000 G
  3. Much of the solution remained so centrifuged for aditional 10 min
  4. Measured the amount left
    1. It was 140 uL so centrifuged again for 10min
  5. And again
  6. Finally DNA solution was reduced to 19 uL so we added 31 uL to make final volume of 50 uL

Electrophoresis

PCR for DNA confirmation
  1. Added 0.4 uL of 6x Sample Buffer to 1 uL of DNA solution and electrophoresed it.
  2. At the same time add added 2 uL of 6x Sample Buffer to 10 uL of flow trough and centrifuged
  • Used marker pUC119/Hinf
  • It was confirmed that DNA was amplified by PCR
    • Between marker bands of 543 bp and 1330 bp PCR product band of 700 bp was visible
  • Due to negligence (electrophoresis for 45 min), flow through with primers flowed through and exited gel
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