Team:HokkaidoU Japan/Notebook/aggregation Week 8

From 2012.igem.org

(Difference between revisions)
Line 10: Line 10:
Single colony isolation of pBAD-RBS-Ag43-dT on pSB1AK3.
Single colony isolation of pBAD-RBS-Ag43-dT on pSB1AK3.
#Picked up one colony.
#Picked up one colony.
-
#Cultivation on LBK(dt,RBS,T7) and LBK(pLacI-RBS-Ag43) for 20 hours.  
+
#Incubated on LBK(dt,RBS,T7) and LBC(pLacI-RBS-Ag43) for 20 hours.  
</p>
</p>
Line 74: Line 74:
[[image:HokkaidoU2012 120820 pT7-RBS on pSB1C3 colop.jpg|thumb|Colony PCR result]]
[[image:HokkaidoU2012 120820 pT7-RBS on pSB1C3 colop.jpg|thumb|Colony PCR result]]
-
The results showed that ligated DNA has 300 ~ 400 bp and the desired products would have 331bp if it were amplified by 100bp up primer and 200bp down primer. Thus we confirmed that pT7-RBS on pSB1C3 was successfully ligated without no.6,9,10 colonies, but these 3 solution were evapolated because of our mistake. We selected No.4 and 5 colony for liquid culture.
+
The results showed that ligated DNA has 300 ~ 400 bp and the desired products would have 331bp if it were amplified by 100bp up primer and 200bp down primer. Thus we confirmed that pT7-RBS on pSB1C3 was successfully ligated without no.6,9,10 colonies, but these 3 solution were evaporated because of our mistake. We selected No.4 and 5 colony for liquid culture.
</p>
</p>
Line 150: Line 150:
Liquid culture for 3 colonies of pBAD-RBS-Ag43-dT on pSB1AK3 (and pT7-RBS on pSB1C3 colony No.4 and 5 which were selected from the results of colony PCR).
Liquid culture for 3 colonies of pBAD-RBS-Ag43-dT on pSB1AK3 (and pT7-RBS on pSB1C3 colony No.4 and 5 which were selected from the results of colony PCR).
#Added 2 ml of LBK (LBC) into culture tubes.
#Added 2 ml of LBK (LBC) into culture tubes.
-
#Resuspended 1 colonies (Resuspended pre-incubated 200ul of LB and colony solution).  
+
#Resuspended 1 colonies.  
#Incubated the tubes at 37C for 16 hours (19 hours).
#Incubated the tubes at 37C for 16 hours (19 hours).
</p>
</p>
Line 158: Line 158:
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==August 21th==
+
==August 21st==
<div>
<div>
==PCR==
==PCR==
<p>
<p>
PCR of pBAD(containing araC)-RBS.
PCR of pBAD(containing araC)-RBS.
-
And, we checked the plasmid which we did mini-prep at August 18th is pBAD-RBS on pSB1A3 or not.
+
And, we checked the plasmid which we refined by mini-prep at August 18th is pBAD-RBS on pSB1A3 or not.
{|class="hokkaidou-table-pcr-reagent"
{|class="hokkaidou-table-pcr-reagent"
Line 237: Line 237:
#Added 1 ml LBK in one culture as a negative control.
#Added 1 ml LBK in one culture as a negative control.
#Added 900 ul LBK and 100 ul 20% L-arabinose.
#Added 900 ul LBK and 100 ul 20% L-arabinose.
-
#Incubated at 37C 130 rpm for 2 hours and 30 minutes.
+
#Incubated at 37C 130 rpm for 2 hrs and 30 min.
-
#Placed tubes on the table at 30 minutes.
+
#Placed tubes on the table at 30 min.
</p>
</p>
==Plasmid extraction==
==Plasmid extraction==
<p>
<p>
-
Mni-prep of pT7-RBS on pSB1C3 of colony No. 4 and 5 selected by the result of colony PCR yesterday. We used mini-prep kit of Nippon genetics: FastGene Plasmid mini kit and finally we eluted the DNA with 20 ul of elution buffer.  
+
Mni-prep of pT7-RBS on pSB1C3 of colony No. 4 and 5 selected by the result of colony PCR at August 20th. We used mini-prep kit of Nippon genetics: FastGene Plasmid mini kit and finally we eluted the DNA with 20 ul of elution buffer.  
Line 249: Line 249:
-
The concentration of 20 ul of mini-prep products were low to digestion or do something so we retry liquid culture of other number of colony solution: No. 1 and No. 2.  
+
The concentration of 20 ul of mini-prep products were too low to digestion or do something so we retry liquid culture of other number of colony solution: No. 1 and No. 2.  
</p>
</p>
Line 257: Line 257:
Liquid culture for Ag43-dT on pSB1AK3 (and pT7-RBS on pSB1C3 colony No. 1 and 2 which were selected from the results of colony PCR).
Liquid culture for Ag43-dT on pSB1AK3 (and pT7-RBS on pSB1C3 colony No. 1 and 2 which were selected from the results of colony PCR).
#Added 2 ml of LBA (LBC) into culture tubes.
#Added 2 ml of LBA (LBC) into culture tubes.
-
#Resuspended 2 colonies (Resuspended pre-incubated 200 ul of LB and colony solution).  
+
#Resuspended 2 colonies.  
#Incubated the tubes at 37C for 18 hours (16 hours).
#Incubated the tubes at 37C for 18 hours (16 hours).
</p>
</p>
Line 265: Line 265:
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==August 22th==
+
==August 22nd==
<div>
<div>
==Plasmid extraction==
==Plasmid extraction==
<p>
<p>
-
Mni-prep of pT7-RBS on pSB1C3 of colony No.1 and 2 selected by the result of colony PCR at 20th. We used mini-prep kit of Nippon genetics: FastGene Plasmid mini kit and finally we eluted the DNA with 20 ul of elution buffer.  
+
Mni-prep of pT7-RBS on pSB1C3 of colony No.1 and 2 selected by the result of colony PCR at August 20th. We used mini-prep kit of Nippon genetics: FastGene Plasmid mini kit and finally we eluted the DNA with 20 ul of elution buffer.  
[[image:HokkaidoU2012 120822 pT7-RBS on pSB1C3 mini-prep No.jpg|thumb|mini-prep result]]
[[image:HokkaidoU2012 120822 pT7-RBS on pSB1C3 mini-prep No.jpg|thumb|mini-prep result]]
Line 275: Line 275:
-
One of Ag43-dT on pSB1AK3 culture did not get myddy. And another one is only a little muddy.
+
One of Ag43-dT on pSB1AK3 culture did not get muddy. And another one is only a little muddy.
-
We tried mini-prep to the latter, we god the 20 ul of DNA solution.
+
We tried mini-prep to the latter, we got the 20 ul of DNA solution.
And then, we did electrophoresis the mini-prep products and (pBAD-RBS and pBAD) gel extract products.
And then, we did electrophoresis the mini-prep products and (pBAD-RBS and pBAD) gel extract products.
Line 359: Line 359:
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==August 23th==
+
==August 23rd==
<div>
<div>
==Digestion==
==Digestion==
Line 435: Line 435:
==Ethanol precipitation==
==Ethanol precipitation==
<p>
<p>
-
Ethanol precipitation to get more high concentration of Ag43-dT on pSB1AK3 solution cut with XbaI & SpeI.
+
Ethanol precipitation to get more high concentration of Ag43-dT on pSB1AK3 solution digested by XbaI and SpeI.
#Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.
#Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.
#Centrifuged at 15000 rpm, 10 min at 4C.
#Centrifuged at 15000 rpm, 10 min at 4C.
Line 445: Line 445:
==Digestion==
==Digestion==
<p>
<p>
-
Digestion to divide Ag43-dT and pSB1AK3 which has same number of bp as Ag43-dT by cut with HindIII.
+
Digestion to divide Ag43-dT and pSB1AK3 which has same number of bp as Ag43-dT by digested by HindIII.
{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
   |-
   |-
Line 451: Line 451:
   |9 ul
   |9 ul
   |-
   |-
-
   |HindIII(15U/ul)
+
   |HindIII(15 U/ul)
   |1 ul
   |1 ul
   |-
   |-
Line 488: Line 488:
-
From this result, we confirmed that the pSB1AK3 was successfully digested with HindIII, but it was not clear how many pSB1AK3 were remaining as non-digested products.
+
From this result, we confirmed that the pSB1AK3 was successfully digested by HindIII, but it could not red how many pSB1AK3 were remaining as non-digested products.
</p>
</p>
Line 503: Line 503:
==Digestion==
==Digestion==
<p>
<p>
-
Digestion of pT7-RBS on pSB1C3 with SpeI, Ag43-dT on pSB1AK3 with EcoRI & XbaI and pBAD-RBS with EcoRI & PstI.
+
Digested pT7-RBS on pSB1C3 by SpeI, and Ag43-dT on pSB1AK3 digested by EcoRI and XbaI, pBAD-RBS digested by EcoRI and PstI.
Ag43-dT on pSB1AK3
Ag43-dT on pSB1AK3
-
 
+
EcoRI/XbaI
-
 
+
-
E&X
+
{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
   |-
   |-
Line 520: Line 518:
   |-
   |-
   |XbaI
   |XbaI
-
   |1
+
   |1 ul
   |-
   |-
   |10xM buffer
   |10xM buffer
Line 534: Line 532:
-
E (control)
+
EcoRI
{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
   |-
   |-
Line 555: Line 553:
-
X (control)
+
XbaI
{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
   |-
   |-
Line 562: Line 560:
   |-
   |-
   |XbaI
   |XbaI
-
   |1
+
   |1 ul
   |-
   |-
   |10xM buffer
   |10xM buffer
Line 576: Line 574:
-
pBAD-RBS(E & S)
+
pBAD-RBS
 +
EcoRI/PstI
{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
   |-
   |-
-
   |DNA solution ( 100ng/ul)
+
   |DNA solution (100 ng/ul)
   |12 ul
   |12 ul
   |-
   |-
Line 586: Line 585:
   |-
   |-
   |SpeI
   |SpeI
-
   |1
+
   |1 ul
   |-
   |-
   |10xH buffer
   |10xH buffer
Line 718: Line 717:
==Transformation==
==Transformation==
<p>
<p>
-
Transformation for ligation product. We used E.coli strain DH5&alpha;.
+
Transformation for ligation product in DH5&alpha;.
#Added 2 ul of DNA to 50 ul of thawed competent cells on ice.
#Added 2 ul of DNA to 50 ul of thawed competent cells on ice.
#Incubated on ice for 30 min.
#Incubated on ice for 30 min.
Line 790: Line 789:
[[image:HokkaidoU2012 120825 coloP pT7-RBS-Ag43-dT on pSB1C3.jpg|thumb|Colony PCR result]]
[[image:HokkaidoU2012 120825 coloP pT7-RBS-Ag43-dT on pSB1C3.jpg|thumb|Colony PCR result]]
-
The results showed that desired DNA were not existed in these picked up colonies. We observed our ethanol precipitation and ligation products electrophoresis result image (see August 24th) then noticed that the concentration of each ethanol precipitation product was reversed. This means vector DNA would be self-ligated by the high ratio of molar in ligation reaction solution. We decided to try the DNA synthesis from digestion reaction of vector DNA once more time.
+
The results showed that desired DNA were not existed in these picked up colonies. We observed our ethanol precipitation and ligation products electrophoresis result image then noticed that the concentration of each ethanol precipitation product was reversed. This means vector DNA would be self-ligated by the high ratio of molar in ligation reaction solution. We decided to try the DNA synthesis from digestion reaction of vector DNA once more time.
</p>
</p>
==Digestion==
==Digestion==
<p>
<p>
-
Digestion of vector DNA: pT7-RBS on pSB1C3 with SpeI. Not to leave the plasmid DNA as plasmid DNA, we cut the DNA for over 18 hrs.  
+
Digest vector DNA: pT7-RBS on pSB1C3 by SpeI. Not to leave the plasmid DNA as plasmid DNA, we digested the DNA for 18 hrs.  
pT7-RBS on pSB1C3 (SpeI)
pT7-RBS on pSB1C3 (SpeI)
{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
   |-
   |-
-
   |DNA solution ( 20ng/ul)
+
   |DNA solution (20 ng/ul)
   |4 ul
   |4 ul
   |-
   |-
Line 845: Line 844:
===Ligation===
===Ligation===
<p>
<p>
-
Ligation of pBAD-RBS + Ag43-dT on pSB1AK3
+
Ligation of pBAD-RBS and Ag43-dT on pSB1AK3
{|class="hokkaidou-table-ligation"
{|class="hokkaidou-table-ligation"
Line 961: Line 960:
==Transformation==
==Transformation==
<p>
<p>
-
Transformation for ligation product. We used E.coli strain DH5&alpha;.
+
Transformation for ligation product in DH5&alpha;.
#Added 2 ul of DNA to 50 ul of thawed competent cells on ice.
#Added 2 ul of DNA to 50 ul of thawed competent cells on ice.
#Incubated on ice for 30 min.
#Incubated on ice for 30 min.

Revision as of 14:10, 26 September 2012

Contents

August 20th

Single colony isolation

Single colony isolation of pBAD-RBS-Ag43-dT on pSB1AK3.

  1. Picked up one colony.
  2. Incubated on LBK(dt,RBS,T7) and LBC(pLacI-RBS-Ag43) for 20 hours.

Colony PCR

Colony PCR to confirm that whether the pT7 and RBS was successfully ligated with pSB1C3 or not.

DNA solution 4 ul
Kapa-Taq(Taq polymerase) 5 ul
Forward Primer(100bp up 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 53.2 30
4 72 60
5 72 60
6 4 HOLD

Cycle:2~4 x 35

We used N1 (DW only) and N2 (pBAD(containing araC)-RBS on pSB1A3) as controls. Desired product is about 300~400bp.

Colony PCR result

The results showed that ligated DNA has 300 ~ 400 bp and the desired products would have 331bp if it were amplified by 100bp up primer and 200bp down primer. Thus we confirmed that pT7-RBS on pSB1C3 was successfully ligated without no.6,9,10 colonies, but these 3 solution were evaporated because of our mistake. We selected No.4 and 5 colony for liquid culture.


PCR

PCR of BBa_I13453 (pBAD only part, it is not contain araC,).

DNA solution 1 ul
KOD-Plus-NEO(Taq polymerase) 1 ul
dNTP 5 ul
MgSO4 3 ul
KOD-Plus-NEO Buffer 5 ul
Forward Primer(Ag43-f4 primer: 10 uM) 1 ul
Reverse Primer(PS-R primer: 10 uM) 1 ul
DW 33 ul
Total 50 ul


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

liquid culture

Liquid culture for 3 colonies of pBAD-RBS-Ag43-dT on pSB1AK3 (and pT7-RBS on pSB1C3 colony No.4 and 5 which were selected from the results of colony PCR).

  1. Added 2 ml of LBK (LBC) into culture tubes.
  2. Resuspended 1 colonies.
  3. Incubated the tubes at 37C for 16 hours (19 hours).


August 21st

PCR

PCR of pBAD(containing araC)-RBS. And, we checked the plasmid which we refined by mini-prep at August 18th is pBAD-RBS on pSB1A3 or not.

DNA solution 1 ul
KOD-Plus-NEO(Taq polymerase) 1 ul
dNTP 5 ul
MgSO4 3 ul
KOD-Plus-NEO Buffer 5 ul
Forward Primer(Ag43-f4 primer: 10 uM) 1 ul
Reverse Primer(PS-R primer: 10 uM) 1 ul
DW 33 ul
Total 50 ul


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

Aggregation check

we cultured the E. coli, which transformed pBAD-RBS-Ag43-dT on pSB1AK3 in LBK. We checked the construct by induction of L-arabinose after 16 hours incubate.

  1. 2 ml of liquid culture divided two culture. (made two 1 ml culture)
  2. Added 1 ml LBK in one culture as a negative control.
  3. Added 900 ul LBK and 100 ul 20% L-arabinose.
  4. Incubated at 37C 130 rpm for 2 hrs and 30 min.
  5. Placed tubes on the table at 30 min.

Plasmid extraction

Mni-prep of pT7-RBS on pSB1C3 of colony No. 4 and 5 selected by the result of colony PCR at August 20th. We used mini-prep kit of Nippon genetics: FastGene Plasmid mini kit and finally we eluted the DNA with 20 ul of elution buffer.


The concentration of 20 ul of mini-prep products were too low to digestion or do something so we retry liquid culture of other number of colony solution: No. 1 and No. 2.

liquid culture

Liquid culture for Ag43-dT on pSB1AK3 (and pT7-RBS on pSB1C3 colony No. 1 and 2 which were selected from the results of colony PCR).

  1. Added 2 ml of LBA (LBC) into culture tubes.
  2. Resuspended 2 colonies.
  3. Incubated the tubes at 37C for 18 hours (16 hours).


August 22nd

Plasmid extraction

Mni-prep of pT7-RBS on pSB1C3 of colony No.1 and 2 selected by the result of colony PCR at August 20th. We used mini-prep kit of Nippon genetics: FastGene Plasmid mini kit and finally we eluted the DNA with 20 ul of elution buffer.

mini-prep result


One of Ag43-dT on pSB1AK3 culture did not get muddy. And another one is only a little muddy. We tried mini-prep to the latter, we got the 20 ul of DNA solution. And then, we did electrophoresis the mini-prep products and (pBAD-RBS and pBAD) gel extract products.

electrophoresis result(1% agarose gel)
electrophoresis result(2% agarose gel)

PCR

PCR of pT7-RBS on pSB1C3.
We used 4 kinds of primer set.
1 : EX-F , PS-R primer
2 : EX-F , 200b down primer
3 : 100b up , PS-R primer
4 : 100b up , 200b down primer
The density of primer solutions is 10 uM.

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


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

Cycle:2~4 x 45


August 23rd

Digestion

Digestion for making pBAD-RBS-Ag43-dT on pSB1AK3. Digestion of pBAD-RBS.

pBAD-RBS (100 ng/ul) 12 ul
Eco RI 1 ul
SpeI 1 ul
10xH buffer 2 ul
DW 4 ul
Total 20 ul

Digestion of Ag43-dT on pSB1AK3.

Ag43-dT (120 ng/ul) 7 ul
Eco RI 1 ul
XbaI 1 ul
10xM buffer 2 ul
DW 9 ul
Total 20 ul


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

Ag43-dT digestion result
pBAD-RBS digestion result

Ethanol precipitation

Ethanol precipitation to get more high concentration of Ag43-dT on pSB1AK3 solution digested by XbaI and SpeI.

  1. Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.
  2. Centrifuged at 15000 rpm, 10 min at 4C.
  3. Removed supernatant and added 220 ul of 70% ethanol.
  4. Centrifuged at 15000 rpm, 10 min at 4C.
  5. Removed supernatant and air drying at room temperature then added 10 ul of DW.

Digestion

Digestion to divide Ag43-dT and pSB1AK3 which has same number of bp as Ag43-dT by digested by HindIII.

DNA solution ( 257ng/ul) 9 ul
HindIII(15 U/ul) 1 ul
10xM buffer 2 ul
DW 8 ul
Total 20 ul


Number Degree Minute
1 37 180
2 70 15
3 4 HOLD


digestion result


From this result, we confirmed that the pSB1AK3 was successfully digested by HindIII, but it could not red how many pSB1AK3 were remaining as non-digested products.

Gel extraction

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


August 24th

Digestion

Digested pT7-RBS on pSB1C3 by SpeI, and Ag43-dT on pSB1AK3 digested by EcoRI and XbaI, pBAD-RBS digested by EcoRI and PstI. Ag43-dT on pSB1AK3 EcoRI/XbaI

DNA solution ( 120ng/ul) 7 ul
EcoRI 1 ul
XbaI 1 ul
10xM buffer 2 ul
DW 9 ul
Total 20 ul


EcoRI

DNA solution ( 120ng/ul) 7 ul
EcoRI 1 ul
10xM buffer 2 ul
DW 10 ul
Total 20 ul


XbaI

DNA solution ( 120ng/ul) 7 ul
XbaI 1 ul
10xM buffer 2 ul
DW 10 ul
Total 20 ul


pBAD-RBS EcoRI/PstI

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


pT7-RBS on pSB1C3 (SpeI)

DNA solution ( 20ng/ul) 4 ul
SpeI 1 ul
10xM buffer 2 ul
DW 13 ul
Total 20 ul


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


pT7-RBS on pSB1C3 digestion result

About pT7-RBS on pSB1C3, we successfully digested the plasmid DNA and converted it to linear DNA.


Gel extraction

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

Ethanol Precipitation

Ethanol precipitation for pT7-RBS on pSB1C3 and Ag43-dT digestion products.

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

Ligation

Ligation for Ag43-dT as insert and pT7-RBS on pSB1C3 as vector. We used Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer.

Vector DNA 4 ul
Insert DNA 4 ul
DW 2 ul
Ligation Mighty Mix 10 ul
Total 20 ul


Ligation reaction time was in detail below.

Degree Minute
16 30
65 10
4 Hold


Ligation result



August 25th

Transformation

Transformation for ligation product in DH5α.

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

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.

Colony PCR result

The results showed that desired DNA were not existed in these picked up colonies. We observed our ethanol precipitation and ligation products electrophoresis result image then noticed that the concentration of each ethanol precipitation product was reversed. This means vector DNA would be self-ligated by the high ratio of molar in ligation reaction solution. We decided to try the DNA synthesis from digestion reaction of vector DNA once more time.

Digestion

Digest vector DNA: pT7-RBS on pSB1C3 by SpeI. Not to leave the plasmid DNA as plasmid DNA, we digested the DNA for 18 hrs. pT7-RBS on pSB1C3 (SpeI)

DNA solution (20 ng/ul) 4 ul
SpeI 1 ul
10xM buffer 2 ul
DW 13 ul
Total 20 ul


Number Degree Minute
1 37 600 (10 hours)
2 60 15
3 4 HOLD


August 26th

Ligation

Ligation of pBAD-RBS and Ag43-dT on pSB1AK3

pBAD-RBS 3 ul
Ag43-dT on pSB1AK3 1 ul
Ligation Mighty Mix(TAKARA) 5 ul
DW 1 ul
Total 10 ul


Degree Minute
16 30
65 10
4 Hold

Transformation

Transformation for ligation product in DH5α.

  1. Added 2 ul of DNA to 50 ul of thawed competent cells on ice.
  2. Incubated on ice for 30 min.
  3. Added 350 ul of LB.
  4. Plated 300 ul of the culture onto first LBA dish and spread.
  5. Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second LBA dish and spread.
  6. Incubated the plates at 37C for 17 hrs and 30 min.


Gel extraction

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

Ethanol Precipitation

Ethanol precipitation for pT7-RBS on pSB1C3 and Ag43-dT digestion products.

  1. Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
  2. Centrifuged at 14000 rpm, 30 min at 4C.
  3. Removed supernatant and added 220 ul of 70% ethanol.
  4. Centrifuged at 15000 rpm, 15 min at 4C.
  5. Removed supernatant and air drying at room temperature then added 5 ul of DW.
Ethanol precipitation result

Ligation

Ligation for Ag43-dT as insert and pT7-RBS on pSB1C3 as vector. We use Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer.

Vector DNA 0.25 ul
Insert DNA 3 ul
DW 1.75 ul
Ligation Mighty Mix 5 ul
Total 20 ul


Ligation reaction time was in detail below.

Degree Minute
16 30
65 10
4 Hold


Transformation

Transformation for ligation product in DH5α.

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