Team:HokkaidoU Japan/Notebook/Week 3

From 2012.igem.org

(Difference between revisions)
(Gel Extraction, Ethanol precipitation)
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==July 16th==
==July 16th==
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
 +
Ag43, dT
===Digestion===
===Digestion===
Results of digestion in 15th.
Results of digestion in 15th.
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#Centrifuged in 15000rpm, 10min at 4C.
#Centrifuged in 15000rpm, 10min at 4C.
#Remove supernatant and air drying in room temperature then added 5ul of DW.
#Remove supernatant and air drying in room temperature then added 5ul of DW.
 +
 +
----
 +
 +
dT(B0015) would be amplified　incorrectly. So we tried another DNA amplification method: PCR. 
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 +
===PCR===
 +
PCR for dT(B0015)
 +
 +
{|class="hokkaidou-table-pcr-reagent"
 +
|-
 +
|DNA solution
 +
|1ul
 +
|-
 +
|KOD-Plus-NEO(Taq polymerase)
 +
|1ul
 +
|-
 +
|dNTP
 +
|5ul
 +
|-
 +
|MgSO4
 +
|3ul
 +
|-
 +
|KOD-Plus-NEO Buffer
 +
|5ul
 +
|-
 +
|Forward Primer(100bp_up forward primer)
 +
|1ul
 +
|-
 +
|Reverse Primer(200bp_down Reverse primer)
 +
|1ul
 +
|-
 +
|DW
 +
|33ul
 +
|-
 +
|Total
 +
|50ul
 +
|}
 +
 +
 +
{|class="hokkaidou-table-pcr-time"
 +
|-
 +
|Number
 +
|Degree
 +
|Second
 +
|-
 +
|1
 +
|94
 +
|120
 +
|-
 +
|2
 +
|98
 +
|10
 +
|-
 +
|3
 +
|58
 +
|30
 +
|-
 +
|4
 +
|68
 +
|30
 +
|-
 +
|5
 +
|4
 +
|HOLD
 +
|}
 +
Cycle:2~4 x 45
 +
 +
 +
 +
[[image:HokkaidoU2012 120716-dt-ethandpcr.jpg|thumb|PCR result image]]
 +
 +
We migrated B0015 mini-prep psoduct, digestion product, and PCR product.
 +
PCRed dT would have 429bp(100bp(added by forward primer) + 129bp(dT) + 200bp(added by reverse primer)). Most bright and thick band in this image has about 300~400 bp. We thought dT was amplified successfully.
 +
 +
 +
----
 +
 +
Ag43
 +
 +
Digestion result of Ag43 was incorrect. We digested Ag43 once more time.
 +
 +
===Digestion===
 +
Digestion for Ag43 with SpeI and PstI.
 +
 +
{|class="hokkaidou-table-digestion"
 +
|-
 +
|Ag43 DNA solution
 +
|9ul
 +
|-
 +
|SpeI
 +
|1ul
 +
|-
 +
|PstI
 +
|1ul
 +
|-
 +
|10xH buffer
 +
|2ul
 +
|-
 +
|DW
 +
|7ul
 +
|-
 +
|Total
 +
|20ul
 +
|}
 +
 +
 +
[[image:|thumb|Digestion result image]]

Revision as of 11:40, 16 July 2012

Contents

July 16th

Ag43, dT

Digestion

Results of digestion in 15th.

Digestion result image

Product:Ag43(K346007)=3120bp and 2070bp, pT7-RBS on pSB1K3=2247bp We confirmed there are some kind of restriction enzyme site in K346007 (digested with SpeI, PstI) and pT7-RBS on pSB1K3 was successfully digested with EcoRI and PstI.　Balance between d+(EcoRI) and d+(E&P:cut with EcoRI and PstI) is about 80bp.


Gel Extraction

Gel Extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics). Got 50ul of DNA solution.


Ethanol Precipitation

Ethanol Precipitation for digestion and gel extraction products.

  1. Added 5ul of NaoAc, 1.5ul of glycogen and 125ul of 100% ethanol.
  2. Centrifuged in 15000rpm, 10min at 4C.
  3. Remove supernatant and added 220ul of 70% ethanol.
  4. Centrifuged in 15000rpm, 10min at 4C.
  5. Remove supernatant and air drying in room temperature then added 5ul of DW.

dT(B0015) would be amplified　incorrectly. So we tried another DNA amplification method: PCR.

PCR

PCR for dT(B0015)

DNA solution 1ul
KOD-Plus-NEO(Taq polymerase) 1ul
dNTP 5ul
MgSO4 3ul
KOD-Plus-NEO Buffer 5ul
Forward Primer(100bp_up forward primer) 1ul
Reverse Primer(200bp_down Reverse primer) 1ul
DW 33ul
Total 50ul


Number Degree Second
1 94 120
2 98 10
3 58 30
4 68 30
5 4 HOLD

Cycle:2~4 x 45


PCR result image

We migrated B0015 mini-prep psoduct, digestion product, and PCR product. PCRed dT would have 429bp(100bp(added by forward primer) + 129bp(dT) + 200bp(added by reverse primer)). Most bright and thick band in this image has about 300~400 bp. We thought dT was amplified successfully.


Ag43

Digestion result of Ag43 was incorrect. We digested Ag43 once more time.

Digestion

Digestion for Ag43 with SpeI and PstI.

Ag43 DNA solution 9ul
SpeI 1ul
PstI 1ul
10xH buffer 2ul
DW 7ul
Total 20ul


[[image:|thumb|Digestion result image]]



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