Team:HokkaidoU Japan/Notebook/aggregation Week 10

From 2012.igem.org

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

Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
Centrifuged at 14000 rpm, 30 min at 4C.
Removed supernatant and added 220 ul of 70% ethanol.
Centrifuged at 15000 rpm, 15 min at 4C.
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

Mixed 2 ul ptet-RBS-eYFP-dT-pSTV28 ligation product to 50 ul of thawed competent cells on ice.
Incubated on ice for 30 min.
Mixed 350 ul of LB.
Incubated for 2 hrs to get the resistance to Chloramphenicol.
Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBC).
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 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

Prepared 2 ml LBA into culture tubes.
Re-suspended 1 colony mixture.
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.

Mixed 2 ul ptet-RBS-eYFP-dT-pSTV28 ligation product to 50 ul of thawed competent cells on ice.
Incubated on ice for 30 min.
Mixed 350 ul of LB.
Incubated for 2 hrs to get the resistance to Chloramphenicol.
Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LB1/2C).
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 19 hrs.