Team:HokkaidoU Japan/Notebook/aggregation Week 7

From 2012.igem.org

(Difference between revisions)
Line 167: Line 167:
Next step, we resuspended these 5 colonies and cultured (add 1800 ul LB and 2 ul Amp) for  hours in 37C.
Next step, we resuspended these 5 colonies and cultured (add 1800 ul LB and 2 ul Amp) for  hours in 37C.
</p>
</p>
 +
 +
==Transformation==
 +
<p>
 +
Transformation of BBa_I13453 (pBAD on pSB1A3) in E. coli(DH5α).
 +
#Added 1 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
 +
#Incubated on ice for 30 min.
 +
#Added 350 ul of LB.
 +
#Prepared and Labeled two petri dishes with LBK.
 +
#Plate 300 ul of the transformation onto first dish and spread.
 +
#Added 450 ul of LB to 50 ul of the transformation and plated 300 ul of it onto second dish and spread.
 +
#Incubated the plates at 37C for 19 hours.
 +
</p>
 +
 +
==Digestion==
 +
<p>
 +
According to sequencing results, we found that we failed to make pT7-RBS on pSB1K3.<br />
 +
So, we tried to 3A assembry again. <br />
 +
When the restriction enzyme digest the DNA, it is important for 3A assembry to digest completly. So, we did activate the restriction enzymes for 10 hours.
 +
 +
pT7 (BBa_I719005)
 +
EcoRI/SpeI
 +
{|class="hokkaidou-table-digestion"
 +
  |-
 +
  |DNA solution (40 ng/ul)
 +
  |12.5 ul
 +
  |-
 +
  |EcoRI
 +
  |1 ul
 +
  |-
 +
  |SpeI
 +
  |1 ul
 +
  |-
 +
  |10xH buffer
 +
  |2 ul
 +
  |-
 +
  |DW
 +
  |3.5 ul
 +
  |-
 +
  |Total
 +
  |20 ul
 +
  |}
 +
 +
RBS (BBa_B0034)
 +
XbaI/PstI
 +
{|class="hokkaidou-table-digestion"
 +
  |-
 +
  |DNA solution (40 ng/ul)
 +
  |12.5 ul
 +
  |-
 +
  |XbaI
 +
  |1 ul
 +
  |-
 +
  |PstI
 +
  |1 ul
 +
  |-
 +
  |10xM buffer
 +
  |2 ul
 +
  |-
 +
  |DW
 +
  |3.5 ul
 +
  |-
 +
  |Total
 +
  |20 ul
 +
  |}
 +
 +
pSB1C3
 +
EcoRI/PstI
 +
{|class="hokkaidou-table-digestion"
 +
  |-
 +
  |DNA solution (25 ng/ul)
 +
  |12 ul
 +
  |-
 +
  |EcoRI
 +
  |1 ul
 +
  |-
 +
  |PstI
 +
  |1 ul
 +
  |-
 +
  |10xH buffer
 +
  |2 ul
 +
  |-
 +
  |DW
 +
  |4 ul
 +
  |-
 +
  |Total
 +
  |20 ul
 +
  |}
 +
</p>
 +
</div><div>
</div><div>
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi -->
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi -->

Revision as of 04:42, 18 August 2012

Contents

August 16th

ligation

We ligated pBad-RBS on pSB1A3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.

pT7-RBS (5 ng/ul) 1 ul
Ag43-dT (25 ng/ul) 2 ul
Ligation Mighty Mix(TAKARA) 5 ul
DW 2 ul
Total 10 ul


Ligation reaction time was written below.

Degree Minute
16 30
65 10
4 Hold

Transformation

Transformation of plasmid DNA ligation product (pT7-RBS-Ag43-dT on pSB1K3) in E. coli(DH5α).

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

Digestion

Ag43-dT on pSB1AK3 digestion with SpeI and XbaI. Ag43-dT SpeI and XbaI

DNA solution (100 ng/ul) 12 ul
SpeI 1 ul
XbaI 1 ul
10xH buffer 2 ul
DW 4 ul
Total 20 ul

pBad-RBS on pSB1A3 electrophoresis result
digestion result

Gel extraction

Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.


August 17th

Colony PCR

Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pBAD-RBS on pSB1A3 or not.

DNA solution 4 ul (1 colony/10 ul DW)
Kapa-Taq(Taq polymerase) 5 ul
Forward Primer(Ag43-f4 primer (50 pmol/ul)) 0.5 ul
Reverse Primer(PS-R primer (50 pmol/ul)) 0.5 ul
Total 10 ul


Number Degree Second
1 95 120
2 95 30
3 53 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 pSB1K3) as controls. Desired product is about 800~1000bp.

Colony PCR result

In this result, N2 has about 500bp band, but N1 has not it. So, we use to liquid culture, No. 7,10,12.
We cultured these DNA in E. coli solution, after added 200 ul LB, for 37C Next step, we resuspended these 5 colonies and cultured (add 1800 ul LB and 2 ul Amp) for hours in 37C.

Transformation

Transformation of BBa_I13453 (pBAD on pSB1A3) in E. coli(DH5α).

  1. Added 1 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
  2. Incubated on ice for 30 min.
  3. Added 350 ul of LB.
  4. Prepared and Labeled two petri dishes with LBK.
  5. Plate 300 ul of the transformation onto first dish and spread.
  6. Added 450 ul of LB to 50 ul of the transformation and plated 300 ul of it onto second dish and spread.
  7. Incubated the plates at 37C for 19 hours.

Digestion

According to sequencing results, we found that we failed to make pT7-RBS on pSB1K3.
So, we tried to 3A assembry again.
When the restriction enzyme digest the DNA, it is important for 3A assembry to digest completly. So, we did activate the restriction enzymes for 10 hours. pT7 (BBa_I719005) EcoRI/SpeI

DNA solution (40 ng/ul) 12.5 ul
EcoRI 1 ul
SpeI 1 ul
10xH buffer 2 ul
DW 3.5 ul
Total 20 ul

RBS (BBa_B0034) XbaI/PstI

DNA solution (40 ng/ul) 12.5 ul
XbaI 1 ul
PstI 1 ul
10xM buffer 2 ul
DW 3.5 ul
Total 20 ul

pSB1C3 EcoRI/PstI

DNA solution (25 ng/ul) 12 ul
EcoRI 1 ul
PstI 1 ul
10xH buffer 2 ul
DW 4 ul
Total 20 ul