Team:HokkaidoU Japan/Notebook/aggregation Week 8

Single colony isolation

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

1. Picked up one colony.
2. Cultivation on LBK(dt,RBS,T7) and LBK(pLacI-RBS-Ag43) in hours.

Colony PCR

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

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 evapolated 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,).

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 (Resuspended pre-cultivated 200ul of LB and colony solution).
3. Incubated the tubes at 37C for 16 hours (19 hours).

PCR

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.

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 negative control.
3. Added 900 ul LBK and 100 ul 20% L-arabinose.
4. Incubated at 37C 130 rpm for 2 hours and 30 minutes.
5. Placed tubes on the table at 30 minutes.

Mini-prep

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.

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.

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 (Resuspended pre-cultivated 200 ul of LB and colony solution).
3. Incubated the tubes at 37C for 18 hours (16 hours).

Mini-prep

Mni-prep of pT7-RBS on pSB1C3 of colony No.1 and 2 selected by the result of colony PCR in 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 myddy. And another one is only a little muddy. We tried mini-prep to the latter, we god 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.

Cycle:2~4 x 45

[[image:|thumb|PCR result]]

Ethanol precipitation

Ethanol precipitation to get more high concentration of Ag43-dT on pSB1AK3 solution cut with XbaI & SpeI.

1. Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.
2. Centrifuged in 15000 rpm, 10 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.

Digestion

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

digestion result

In 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.

Gel extraction

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

Digestion

Digestion of pT7-RBS on pSB1C3 with SpeI, Ag43-dT on pSB1AK3 with EcoRI & XbaI and pBAD-RBS with EcoRI & PstI. Ag43-dT on pSB1AK3 E&X

E (control)

X (control)

pBAD-RBS(E & S)

pT7-RBS on pSB1C3 (SpeI)