Team:HokkaidoU Japan/Notebook/aggregation Week 10

From 2012.igem.org

(Difference between revisions)
Line 277: Line 277:
#Removed supernatant and added 220 ul of 70% ethanol.
#Removed supernatant and added 220 ul of 70% ethanol.
#Centrifuged at 15000 rpm, 10 min at 4C.
#Centrifuged at 15000 rpm, 10 min at 4C.
-
#Removed supernatant and air drying at room temperature then added 5 ul of DW.  
+
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW.  
Line 598: Line 598:
#Removed supernatant and added 220 ul of 70% ethanol.
#Removed supernatant and added 220 ul of 70% ethanol.
#Centrifuged at 15000 rpm, 15 min at 4C.
#Centrifuged at 15000 rpm, 15 min at 4C.
-
#Removed supernatant and air drying at room temperature then added 5 ul of DW.  
+
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW.  
Line 654: Line 654:
#Plated 300 ul of the culture onto first dish and spread.
#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.
#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 19 hours.
+
#Incubated the plates at 37C for 19 hrs.
</p>
</p>
Line 739: Line 739:
#Plated 300 ul of the culture onto first dish and spread.
#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.
#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 19 hours.
+
#Incubated the plates at 37C for 19 hrs.
</p>
</p>
</div></div>
</div></div>

Revision as of 21:30, 26 September 2012

Contents

September 3rd

Colony PCR

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

DNA solution 4 ul
Kapa-Taq(Taq polymerase) 10 ul
Forward Primer(Ag43-f4) 1 ul
Reverse Primer(PS-Reverse) 1 ul
DW 4 ul
Total 20 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-RBS-Ag43-dT on pSB1Ak3) as controls. Desired product is about 600bp.

Colony PCR result

Colony PCR of eCFP-RBS-pSB1A2

DNA solution 4 ul
Kapa-Taq(Taq polymerase) 5 ul
Forward Primer(EX-F primer) 0.5 ul
Reverse Primer(PS-R down primer) 0.5 ul
Total 10 ul


Number Degree Second
1 95 120
2 95 30
3 68.9 30
4 72 60
5 72 60
6 4 HOLD

Cycle:2~4 x 35

We used N1 (DW only) and N2 (ptetR-RBS-eCFP-dT-pSB1A2) as controls. Desired product is about 776bp.

Colony PCR result


We confirmed that about 70% of ligated DNA formed our desired construct. We selected No. 2 and 5 colony for incubation and store No. 7 and 8 colony mixture at 4C.

Incubation of eCFP-RBS-pSB1A2 for plasmid extraction

  1. Prepared 2 ml LBA into culture tubes.
  2. Re-suspended 2 colony mixture (No. 2 and No. 5 respectively).
  3. Incubated at 37C for 17 hrs.


Estimation of concentration of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2

No. 2,5 means the colony number of colony PCR of eCFP-RBS-pSB1A2.

electrophoresis result


We estimated the concentration of eCFP-RBS-pSB1A2 is 30 ng/ul and pBAD-RBS-pSB1A2 is 50 ng/ul.


Digestion of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2

To make a construct of pBAD-RBS-eCFP-RBS-pSB1A2, we digested eCFP-RBS-pSB1A2 with XbaI & PstI and pBAD-RBS-pSB1A2 with SpeI and PstI. And we digested eCFP-pSB1A2 with XbaI and SpeI as a control for confirmation of the ability to digest. Insert (eCFP-RBS-pSB1A2)

DNA solution (30 ng/ul) 22 ul
XbaI 1 ul
PstI 1 ul
10xM buffer 3 ul
DW 3 ul
Total 30 ul



Vector(pBAD-RBS-pSB1A2)

DNA solution (50 ng/ul) 3 ul
SpeI 1 ul
PstI 1 ul
10xH buffer 2 ul
DW 13 ul
Total 20 ul


control (eCFP-pSB1A2)

DNA solution (30 ng/ul) 5 ul
XbaI 1 ul
SpeI 1 ul
10xM buffer 2 ul
DW 11 ul
Total 20 ul


Number Degree Minute
1 37 120
2 60 15
3 4 HOLD


digestion result


From this image, we confirmed that DNA were digested into fragments and all of restriction enzyme worked.


September 4th

Plasmid extraction

Plasmid extraction of pBAD-RBS-Ag43-dT on pSB1C3. We used plasmid extraction kit of Nippon genetics: FastGene Plasmid mini kit and finally we eluted the DNA with 50 ul of elution buffer.

Plasmid extraction result

Ethanol precipitation of digestion products (eCFP-RBS and pBAD-RBS-pSB1A2) and estimation of concentration

  1. Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
  2. Centrifuged at 15000 rpm, 15 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 dried out at room temperature, after that added 10 ul of DW.
ethanol precipitation result

We estimated the concentration of ethanol presipitation products.The concentration of Insert DNA solution is about 20 ng/ul and Vector DNA solution is about 50 ng/ul.

Ligation of eCFP-RBS and pBAD-RBS-pSB1A2

Vector DNA (50 ng/ul) 1.5 ul
Insert DNA (20 ng/ul) 4 ul
Ligation Mighty Mix 6 ul
DW 0.5 ul
Total 12 ul


Ligation reaction time was in detail below.

Degree Minute
16 30
65 10
4 Hold

Transformation of pBAD-RBS-eCFP-RBS-pSB1A2

  1. Mixed 2 ul pBAD-RBS-eCFP-RBS-pSB1A2 ligation product 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 LB plate medium which contained appropriate antibiotics (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 25 hours.


Digestion of ptet-RBS-eYFP-dT-pSB1A2 and pSTV28

To make a construct of ptet-RBS-eYFP-dT-pSTV28, we digested ptet-RBS-eYFP-dT-pSB1A2 with EcoRI and PstI, and pSTV28 with EcoRI and PstI. And we digested Ag43-dT-pSB1AK3 by HindIII. Insert (ptet-RBS-eYFP-dT-pSB1A2)

DNA solution (30 ng/ul) 20 ul
EcoRI 1 ul
PstI 1 ul
10xH buffer 3 ul
DW 5 ul
Total 30 ul



Vector(pSTV28)

DNA solution (50 ng/ul) 3 ul
EcoRI 1 ul
PstI 1 ul
10xH buffer 2 ul
DW 13 ul
Total 20 ul


Ag43-dT-pSB1AK3

DNA solution (30 ng/ul) 30 ul
HindIII 2 ul
10xM buffer 5 ul
DW 13 ul
Total 50 ul


Number Degree Minute
1 37 120
2 60 15
3 4 HOLD


digestion result


We confirmed that ptet-RBS-eYFP-dT-pSB1A2 was partially digested and pSTV28 would be successfully digested we think. But we couldn't confirm whether Ag43-dT-pSB1AK3 was digested or not. We did a gel-extraction of these digested DNA and got 50ul solution of each DNA.

September 5th

Digestion

We digested plasmid extraction products (pBAD-RBS-Ag43-dT on pSB1AK3) by restriction enzyme. Because I'd like to check the size of insert and vector of getting plasmid. No. 1

DNA solution 1 ul
EcoRI 1 ul
10xH buffer 1 ul
DW 7 ul
Total 10 ul

No. 2

DNA solution 1 ul
XbaI 1 ul
10xM buffer 1 ul
10xBSA buffer 1 ul
DW 6 ul
Total 10 ul

No. 3

DNA solution 1 ul
SpeI 1 ul
10xM buffer 1 ul
DW 7 ul
Total 10 ul

No. 4

DNA solution 1 ul
PstI 1 ul
10xH buffer 1 ul
DW 7 ul
Total 10 ul

No. 5

DNA solution 2 ul
EcoRI 1 ul
SpeI 1 ul
10xH buffer 2 ul
DW 14 ul
Total 20 ul


Number Degree Minute
1 37 120
2 60 15
3 4 HOLD



Digestion resulsts


September 6th

Ag43 expressing

We usually used plastic tube for incubating. The E. coli expressing Ag43 attachment to the face of tube wall.

plastic incubated results

We estimated the reason is attachment to hydrophobic materials. We incubated in 1 ml of LBA (one contained 1% of L-arabinose, another did not contain L-arabinose) at 37C in glass tubes.

Ethanol precipitation of ptet-RBS-eYFP-dT-pSB1A2 and pSTV28

  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 dried out at room temperature, after that added 10 ul of DW.
ethanol precipitation result

We confirmed that the concentration of Insert DNA solution is 10~20 ng/ul and Vector DNA solution is 30~40 ng/ul.

Ligation of ptet-RBS-eYFP-dT-pSB1A2 and pSTV28

Vector DNA (30~40 ng/ul) 1.5 ul
Insert DNA (10~20 ng/ul) 4 ul
Ligation Mighty Mix 6 ul
DW 0.5 ul
Total 12 ul


Ligation reaction time was in detail below.

Degree Minute
16 30
65 10
4 Hold

Transformation of ptet-RBS-eYFP-dT-pSTV28

  1. Mixed 2 ul ptet-RBS-eYFP-dT-pSTV28 ligation product to 50 ul of thawed competent cells on ice.
  2. Incubated on ice for 30 min.
  3. Mixed 350 ul of LB.
  4. Incubated for 2 hrs to get the resistance to Chloramphenicol.
  5. Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBC).
  6. Plated 300 ul of the culture onto first dish and spread.
  7. Mixed 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 19 hrs.


Colony PCR of pBAD-RBS-eCFP-RBS-pSB1A2

DNA solution 4 ul
Kapa-Taq(Taq polymerase) 5 ul
Forward Primer(pbad-f2 primer) 0.5 ul
Reverse Primer(PS-R down primer) 0.5 ul
Total 10 ul


Number Degree Second
1 95 120
2 95 30
3 53.3 30
4 72 60
5 72 60
6 4 HOLD

Cycle:2~4 x 35

We used N1 (DW only) as controls. Desired product is about 900bp.

Colony PCR result


We thought that almost all of ligated DNA successfully ligated to our desired construct. We selected No. 1 colony for incubation and store No. 2,3 and 4 colony liquid medium at 4C.

Incubation of pBAD-RBS-eCFP-RBS-pSB1A2 for plasmid extraction

  1. Prepared 2 ml LBA into culture tubes.
  2. Re-suspended 1 colony mixture.
  3. Incubated at 37C for 18 hrs.

Transformation of ptet-RBS-eYFP-dT-pSTV28 and Test to confirm appropriate antibiotic amount for screening of pSTV28

To confirm how amount of chloramphenicol is needed for screening pSTV28 (Low copy plasmid) , we plated transformed cells on the special LBC which contains half amount of chloramphenicol we usually add. Now we call this LBC plate medium as LB1/2C.

  1. Mixed 2 ul ptet-RBS-eYFP-dT-pSTV28 ligation product to 50 ul of thawed competent cells on ice.
  2. Incubated on ice for 30 min.
  3. Mixed 350 ul of LB.
  4. Incubated for 2 hrs to get the resistance to Chloramphenicol.
  5. Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LB1/2C).
  6. Plated 300 ul of the culture onto first dish and spread.
  7. Mixed 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 19 hrs.


September 7th

Estimation of concentration of pBAD-RBS-eCFP-RBS-pSB1A2

electrophoresis result


We estimated the concentration of pBAD-RBS-eCFP-RBS-pSB1A2 is about 30 ng/ul.

Incubation of ptet-RBS-eYFP-dT-pSTV28 for plasmid extraction

  1. Prepared 5~6 ml LBC into culture tubes.
  2. Re-suspended 2 colony.
  3. Incubated at 37C for 7 hrs.
Plasmid extraction result


Retrieved from "http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_10"