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Team:HokkaidoU Japan/Notebook/August13
From 2010.igem.org
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(→ミニプレで増えなかったパーツのPCR) |
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|style="text-align:left;"| 1 uL | |style="text-align:left;"| 1 uL | ||
|- | |- | ||
| - | |style="text-align:right;"| Template (1-18F) : | + | |style="text-align:right;"| Template ([[Team:HokkaidoU_Japan/Parts#BioBricks|1-18F]]) : |
|style="text-align:left;"| 1 uL | |style="text-align:left;"| 1 uL | ||
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==Removal of Primers== | ==Removal of Primers== | ||
# Added 150 uL of TE to 50 uL of PCR product, each | # Added 150 uL of TE to 50 uL of PCR product, each | ||
| - | # Transfered into Microcon YM-10 filter and cetrifuged for 20 min at | + | # Transfered into Microcon YM-10 filter and cetrifuged for 20 min at 14,000 G |
# Much of the solution remained so centrifuged for aditional 10 min | # Much of the solution remained so centrifuged for aditional 10 min | ||
# Measured the amount left | # Measured the amount left | ||
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==Electrophoresis== | ==Electrophoresis== | ||
| - | [[Image:HokkaidoU Japan 20100813a.jpg|200px|right|thumb|]] | + | [[Image:HokkaidoU Japan 20100813a.jpg|200px|right|thumb|PCR for DNA confirmation]] |
# Added 0.4 uL of 6x Sample Buffer to 1 uL of DNA solution and electrophoresed it. | # Added 0.4 uL of 6x Sample Buffer to 1 uL of DNA solution and electrophoresed it. | ||
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* It was confirmed that DNA was amplified by PCR | * 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 | ** Between marker bands of 543 bp and 1330 bp PCR product band of 700 bp was visible | ||
| - | * Due to negligence (electrophoresis for | + | * Due to negligence (electrophoresis for 45 min), flow through with primers flowed through and exited gel |
<div style="clear:both"> | <div style="clear:both"> | ||
Latest revision as of 07:26, 27 October 2010
since 13 Jul 2010
since 1 Aug 2010
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
- Added 150 uL of TE to 50 uL of PCR product, each
- Transfered into Microcon YM-10 filter and cetrifuged for 20 min at 14,000 G
- Much of the solution remained so centrifuged for aditional 10 min
- Measured the amount left
- It was 140 uL so centrifuged again for 10min
- And again
- Finally DNA solution was reduced to 19 uL so we added 31 uL to make final volume of 50 uL
Electrophoresis
- Added 0.4 uL of 6x Sample Buffer to 1 uL of DNA solution and electrophoresed it.
- 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
