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. 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.

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,).

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).

PCR

PCR of pBAD(containing araC)-RBS. And, we checked the plasmid which we refined by plasmid extraction 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 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 Plasmid extraction 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 plasmid extraction 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).

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 plasmid extraction kit of Nippon genetics: FastGene Plasmid mini kit and finally we eluted the DNA with 20 ul of elution buffer.

plasmid extraction result

One of Ag43-dT on pSB1AK3 culture did not get muddy. And another one is only a little muddy. We tried plasmid extraction to the latter, we got the 20 ul of DNA solution. And then, we did electrophoresis the plasmid extraction 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

Digestion

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

Digestion of Ag43-dT on pSB1AK3.

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 dried out at room temperature, after that 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.

digestion result

From this result, we confirmed that the pSB1AK3 was successfully digested by HindIII, but it could not confirm how many pSB1AK3 were remained 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

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

EcoRI

XbaI

pBAD-RBS
EcoRI/PstI

pT7-RBS on pSB1C3 (SpeI)

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 dried out at room temperature, after that added 10 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.

Ligation reaction time was in detail below.

Ligation result

Transformation

Transformation for ligation product into 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.

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 electrophoresis result image of ligation products, 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 ligated DNA 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)

Ligation

Ligation of pBAD-RBS and Ag43-dT on pSB1AK3

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 dried out at room temperature, after that added 10 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.

Ligation reaction time was in detail below.

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 18 hrs.