Team:HokkaidoU Japan/Notebook/aggregation Week 9

From 2012.igem.org

(Difference between revisions)
(28th)
(28th ~ptet-RBs-eYFP-dT transformation)
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|}
|}
Cycle:2~4 x 35
Cycle:2~4 x 35
 +
 +
[[image:|thumb|PCR result]]
</p>
</p>
Line 163: Line 165:
<div>
<div>
==mini-prep of pT7-RBS-Ag43-dT on pSB1C3==
==mini-prep of pT7-RBS-Ag43-dT on pSB1C3==
-
Mini-prep of pT7-RBS-Ag43-dT on pSB1C3
 
<p>
<p>
 +
Mini-prep of pT7-RBS-Ag43-dT on pSB1C3. We re-suspended No.1,2 colonies and incubated.
[[image:|thumb|mini-prep result]]
[[image:|thumb|mini-prep result]]
</p>
</p>
 +
 +
==Estimation of Concentration of RBS-eYFP-dT and ptetR-pSB1A2==
 +
<p>
 +
{|
 +
|Solution
 +
|Value(ul)
 +
|-
 +
|1kb ladder
 +
|1
 +
|-
 +
|Insert
 +
|1
 +
|-
 +
|Vector
 +
|1
 +
|-
 +
|}
 +
 +
 +
[[image:|thumb|Electrophoresis result]]
 +
 +
 +
From this result, We estimated that the concentration of Insert DNA solution is about 40mM, and Vector DNA is also about 40mM.
 +
</p>
 +
 +
==Digestion of ptet-pSB1A2 and RBS-Ag43-dT==
 +
<p>
 +
ptet-pSB1A2(Vector) was digested with SpeI and PstI, and RBS-Ag43-dT(Insert) was cut with XbaI and PstI.
 +
 +
 +
 +
 +
Vector
 +
{|class="hokkaidou-table-digestion"
 +
  |-
 +
  |DNA solution ( 40ng/ul)
 +
  |5 ul
 +
  |-
 +
  |SpeI
 +
  |0.5 ul
 +
  |-
 +
  |PstI
 +
  |0.5 ul
 +
  |-
 +
  |10xH buffer
 +
  |2 ul
 +
  |-
 +
  |DW
 +
  |12 ul
 +
  |-
 +
  |Total
 +
  |20 ul
 +
  |}
 +
 +
 +
 +
 +
Inset
 +
{|class="hokkaidou-table-digestion"
 +
  |-
 +
  |DNA solution ( 40ng/ul)
 +
  |14 ul
 +
  |-
 +
  |XbaI
 +
  |1 ul
 +
  |-
 +
  |PstI
 +
  |1 ul
 +
  |-
 +
  |10xM buffer
 +
  |2 ul
 +
  |-
 +
  |DW
 +
  |2 ul
 +
  |-
 +
  |Total
 +
  |20 ul
 +
  |}
 +
 +
 +
 +
{|class="hokkaidou-table-digestion"
 +
|-
 +
|Number
 +
|Degree
 +
|Minute
 +
|-
 +
|1
 +
|37
 +
|180
 +
|-
 +
|2
 +
|60
 +
|15
 +
|-
 +
|3
 +
|4
 +
|HOLD
 +
|}
 +
 +
 +
[[image:|thumb|digestion result]]
 +
 +
From this result, we confirmed that Insert and Vector DNA were digested.
 +
</p>
 +
 +
 +
==Ethanol Precipitation==
 +
<p>
 +
Ethanol precipitation for ptet-pSB1A2 and RBS-eYFP-dT digestion products.
 +
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
 +
#Centrifuged in 15000 rpm, 15 min at 4C.
 +
#Remove supernatant and added 220 ul of 70% ethanol.
 +
#Centrifuged in 15000 rpm, 10 min at 4C.
 +
#Remove supernatant and air drying in room temperature then added 10 ul of DW.
 +
</p>
 +
==Ligation==
 +
<p>
 +
Ligation for ptet-pSB1A2 and RBS-eYFP-dT.
 +
We used Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer.
 +
 +
 +
{|class="hokkaidou-table-ligation"
 +
|-
 +
|Vector DNA
 +
|3 ul
 +
|-
 +
|Insert DNA
 +
|3 ul
 +
|-
 +
|Ligation Mighty Mix
 +
|6 ul
 +
|-
 +
|Total
 +
|12 ul
 +
|}
 +
 +
 +
Ligation reaction time was in detail below.
 +
 +
{|class="hokkaidou-table-ligation"
 +
|-
 +
|Degree
 +
|Minute
 +
|-
 +
|16
 +
|30
 +
|-
 +
|65
 +
|10
 +
|-
 +
|4
 +
|Hold
 +
|}
 +
</p>
 +
 +
==Transformation==
 +
<p>
 +
Transformation for ligation product. We used E.coli strain DH5&alpha;.
 +
#Mixed 1 ul of DNA to 50 ul of thawed competent cells on ice.
 +
#Incubated on ice for 30 min.
 +
#Mixed 350 ul of LB. 
 +
#Prepared and Labeled two plastic plates with LBA.
 +
#Plated 300 ul of the culture onto first dish and spread.
 +
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.
 +
#Incubated the plates at 37C for 15 hours.
 +
</p>
 +
</div></div>
</div></div>

Revision as of 07:34, 29 August 2012

Contents

August 27th

Colony PCR

Colony PCR to confirm that whether the pT7-RBS-Ag43-dT on pSB1C3 was successfully ligated or not.

DNA solution 4 ul
Kapa-Taq(Taq polymerase) 5 ul
Forward Primer(ag43-f4 primer) 0.5 ul
Reverse Primer(200bp down primer) 0.5 ul
Total 10 ul


Number Degree Second
1 95 120
2 95 30
3 53.0 30
4 72 60
5 72 60
6 4 HOLD

Cycle:2~4 x 35

We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls. Desired product is about 695bp.

[[image:|thumb|Colony PCR result]]



Incubation for mini-prep of pT7-RBS-Ag43-dT on pSB1C3

Incubation of pT7-RBS-Ag43-dT on pSB1C3 in LBC liquid medium.

  1. Prepared 2 ml LBC into culture tubes.
  2. Re-suspended 2 colonies (No.1 and No.2 respectively).
  3. Incubated at 37C for 15 hrs.

Transformation of ptet-RBS(B0034)-CFP-dT on pSB1A2

  1. Mixed 1 ul DNA to 50 ul of thawed competent cells on ice.
  2. Incubated on ice for 30 min.
  3. Mixed 350 ul of LB.
  4. Prepared and Labeled two plastic plates with LBC.
  5. Plated 300 ul of the culture onto first dish and spread.
  6. Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.
  7. Incubated the plates at 37C for 15 hours.

PCR of RBS-YFP-dT

Amplified the construct by 100bp-up-EX primer and 200bp-down-PS primer. Mixed PCR solutions.

Solution Volume(ul)
DNA 1
100bp-up-EX 1
200bp-down-PS 1
MgSO4 3
dNTP 5
10x KOD-Plus-Neo Buffer 5
KOD-Plus-Neo 1
DW 33
Total 50
Number Degree Second
1 94 120
2 98 10
3 58.2 30
4 68 60
5 4 HOLD

Cycle:2~4 x 35

[[image:|thumb|PCR result]]


August 28th

mini-prep of pT7-RBS-Ag43-dT on pSB1C3

Mini-prep of pT7-RBS-Ag43-dT on pSB1C3. We re-suspended No.1,2 colonies and incubated. [[image:|thumb|mini-prep result]]

Estimation of Concentration of RBS-eYFP-dT and ptetR-pSB1A2

Solution Value(ul)
1kb ladder 1
Insert 1
Vector 1


[[image:|thumb|Electrophoresis result]]


From this result, We estimated that the concentration of Insert DNA solution is about 40mM, and Vector DNA is also about 40mM.

Digestion of ptet-pSB1A2 and RBS-Ag43-dT

ptet-pSB1A2(Vector) was digested with SpeI and PstI, and RBS-Ag43-dT(Insert) was cut with XbaI and PstI. Vector

DNA solution ( 40ng/ul) 5 ul
SpeI 0.5 ul
PstI 0.5 ul
10xH buffer 2 ul
DW 12 ul
Total 20 ul



Inset

DNA solution ( 40ng/ul) 14 ul
XbaI 1 ul
PstI 1 ul
10xM buffer 2 ul
DW 2 ul
Total 20 ul


Number Degree Minute
1 37 180
2 60 15
3 4 HOLD


[[image:|thumb|digestion result]]

From this result, we confirmed that Insert and Vector DNA were digested.


Ethanol Precipitation

Ethanol precipitation for ptet-pSB1A2 and RBS-eYFP-dT digestion products.

  1. Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
  2. Centrifuged in 15000 rpm, 15 min at 4C.
  3. Remove supernatant and added 220 ul of 70% ethanol.
  4. Centrifuged in 15000 rpm, 10 min at 4C.
  5. Remove supernatant and air drying in room temperature then added 10 ul of DW.

Ligation

Ligation for ptet-pSB1A2 and RBS-eYFP-dT. We used Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer.

Vector DNA 3 ul
Insert DNA 3 ul
Ligation Mighty Mix 6 ul
Total 12 ul


Ligation reaction time was in detail below.

Degree Minute
16 30
65 10
4 Hold

Transformation

Transformation for ligation product. We used E.coli strain DH5α.

  1. Mixed 1 ul of DNA to 50 ul of thawed competent cells on ice.
  2. Incubated on ice for 30 min.
  3. Mixed 350 ul of LB.
  4. Prepared and Labeled two plastic plates with LBA.
  5. Plated 300 ul of the culture onto first dish and spread.
  6. Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.
  7. Incubated the plates at 37C for 15 hours.