Team:HokkaidoU Japan/Notebook/aggregation Week 9

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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.

Colony PCR result


The result did not show the band clearly. We selected No.1 and 2 colony for incubation.



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 with 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



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.

mini-prep result

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

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


Electrophoresis result


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

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


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 (ptet-RBs-eYFP-dT on pSB1A2). 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.


Gel extraction product check of pT7-RBS-Ag43-dT on pSB1C3

We couldn't get desired plasmid DNA by transformation. We doubted contamination of non-digested vector DNA and decided to test the gel extraction product of pT7-RBS on pSB1C3 was successfully separated from non-digested product or not by transformation.

  1. Mixed 1 ul of each DNA of ligation product and digestion 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 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 16 hours.
result:There were no colony on the LBC plate that was spread solution mixed digestion product.


August 29th

PCR of eCFP(E0020)

Amplified the part with 100bp-up-EX primer and 200bp-down-PS primer. Desired product is about 800~900bp. 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

Colony PCR

Colony PCR of pT7-RBS-Ag43-dT on pSB1C3 transformed in 28th.

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. This length is almost same as N2.

Colony PCR result


We decided to incubate the No.7 and 8 colony.


Incubation for mini-prep of pT7-RBS-Ag43-dTonpSB1C3 and Ag43-dTonpSB1AK3

Incubation of pT7-RBS-Ag43-dTonpSB1C3 (and Ag43-dT on pSB1AK3) in LBC (LBA) liquid medium.

  1. Prepared 2 ml LBC (LBA) into culture tubes.
  2. Re-suspended 2 colonies No.7 and No.8 respectively. Ag43-dT on pSB1AK3 was re-suspended the N2 colony.
  3. Incubated at 37C for hrs.


Estimation of Concentration of RBS-eYFP-dT (PCR product) and ptetR-pSB1A2

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


Electrophoresis result


From this result, We estimated that the concentration of Insert DNA solution is about 36ng/ul, and Vector DNA is also about 29ng/ul.

Digestion of ptet-pSB1A2 and RBS-Ag43-dT

ptet-pSB1A2(Vector) was digested with EcoRI and SpeI, and RBS-Ag43-dT(Insert) was cut with EcoRI and XbaI. And we used construct of pT7-RBS on pSB1C3 as control for the function of XbaI. Insert (eCFP)

DNA solution ( 36ng/ul) 5 ul
EcoRI 1 ul
SpeI 1 ul
10xH buffer 2 ul
DW 5 ul
Total 20 ul



Vector(RBS on pSB1A2)

DNA solution ( 29ng/ul) 6 ul
EcoRI 1 ul
XbaI 1 ul
10xM buffer 2 ul
DW 10 ul
Total 20 ul


control (pT7-RBs on pSB1C3)

DNA solution (30 ng/ul) 6 ul
XbaI 1 ul
10xM buffer 2 ul
100x BSA 0.2
DW 11 ul
Total 20 ul


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


digestion result

From this result, Vector DNA were digested with XbaI, but remains some amount of non-digested DNA. And about Insert DNA, the DNA solution were not existed in both d- and d+ lines. This means we failed to extract from gel after electrophoresis. We decided to retry PCR.

PCR of eCFP(E0020)

Amplified the part with 100bp-up-EX primer and 200bp-down-PS primer. Desired product is about 800~900bp. 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 58.0 30
4 68 60
5 4 HOLD

Cycle:2~4 x 35


August 30th

Estimation of concentration of eCFP, pT7-RBS-Ag43-dT-pSB1C3 and Ag43-dT-pSB1AK3

No.7,8 means the colony number of colony PCR of pT7-RBS-Ag43-dT-pSB1C3. [[image:|thumb|electrophoresis result]] We failed the PCR for E0020. We estimated the concentration of pT7-RBS-Ag43-dT-pSB1C3 is 31 ng/ul and Ag43-dT-pSB1AK3 is 35ng/ul.