Team:HokkaidoU Japan/Notebook/Week 3

Ag43, dT

Digestion

Results of digestion in 15th.

Digestion result image

Product:Ag43(K346007)=3120bp and 2070bp, pT7-RBS on pSB1K3=2247bp We confirmed there are some kind of restriction enzyme site in K346007 (digested with SpeI, PstI) and pT7-RBS on pSB1K3 was successfully digested with EcoRI and PstI.　Balance between d+(EcoRI) and d+(E&P:cut with EcoRI and PstI) is about 80bp.

Gel Extraction

Gel Extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics). Got 50ul of DNA solution.

Ethanol Precipitation

Ethanol Precipitation for digestion and gel extraction products.

1. Added 5ul of NaoAc, 1.5ul of glycogen and 125ul of 100% ethanol.
2. Centrifuged in 15000rpm, 10min at 4C.
3. Remove supernatant and added 220ul of 70% ethanol.
4. Centrifuged in 15000rpm, 10min at 4C.
5. Remove supernatant and air drying in room temperature then added 5ul of DW.

dT(B0015) would be amplified　incorrectly and couldn't get enough digestion product. So we tried another DNA amplification method: PCR then digested.

PCR

PCR for dT(B0015)

Cycle:2~4 x 45

PCR result image

We migrated B0015 mini-prep psoduct, digestion product, and PCR product. PCRed dT would have 429bp(100bp(added by forward primer) + 129bp(dT) + 200bp(added by reverse primer)). Most bright and thick band in this image has about 300~400 bp. We thought dT was amplified successfully.

Gel extraction

Gel Extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics). Got 50ul of DNA solution.

Digestion

Digestion for dT which amplified with PCR. Digested with XbaI and PstI. dT

Ag43

Digestion result of Ag43 was incorrect. We digested Ag43 once more time.

Digestion

Digestion for Ag43 with SpeI and PstI.

Digestion result image

There are same results with digestion result of recent. We thought PstI would cut different site. What is this 500bp fragment????

Gel extraction

Gel Extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics). Got 50ul of DNA solution.

Liquid Culture

Liquid culture for single colony isolated pT7-RBS on pSB1C3 and Ag43-dT on pSB1T3. Ag43-dT on pSB1T3 colonies were closely existed so we would picked up two or more colonies.

1. Picked up one (or tow?) colony from single colony isolated plates　by platinum loop.
2. Dipped into 2ml of LBC and LBT.
3. Cultivated.

Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3

Mini-prep

Mini-prep for Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3 liquid culture products cultivated from yesterday(16th). We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50ul of DNA solutions.

Electrophoresis

Electrophoresis for digestion product of K346007(Ag43) with EcoRI and SpeI as a reference to confirm SpeI cuts correct site or not and PstI didn't work correctly(in past experiments). And mini-prep products of Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3 also migrated. If digestion were succeeded, there are two bands would be seen: about 3120bp and 2070bp. And if ligation-transformation-mini-prep were succeeded, there would be existed two bands in each lanes: about 5000bp and 2000bp.

Digestion and mini-prep result image

In this result we confirmed Ag43 was successfully digested with EcoRI and SpeI, and there were no other extra bands which had been existed double digested with EcoRI & PstI and SpeI & PstI. would PstI not work correctly?

We planed to use dT as vector and Ag43 as Insert. A problem is if dT on pSB1AK3 is used as vector and Ag43 used as insert, we can't select correct Ag43 band in gel extraction phase because DNA bp of Ag43 is nearly same as pSB1AK3. Thus we tried to use dT on pSB1T3 as vector and Ag43 on pSB1C3 as vector and ligate with standard assembly.

About mini-prep products, in Ag43-dT on pSB1T3, there were two plasmid DNA bands about 8000bp and 3000bp in one lane. We thought which means we failed single colony isolation then resuspended two another E.coli colonies another ligated DNA were transformed. In pT7-RBS on pSB1C3, we needed about 2000bp plasmid DNA and there were about 1500bp of DNA. Plasmid DNA migrates more far than linear DNA so we thought we got correct DNA.

To confirm mini-prep products were really ligated correct DNA fragments, first we gel extracted Ag43-dT on pSB1T3(low bp band) and digested with EcoRI and PstI.

dT Vector plasmid change To use dT as an vector, we needed to change plasmid backbone pAB1AK3 to pSB1T3.

Digestion

Digestion to change the plasmid backbone. Used DNA solution as PCR product(done in 16th) and digestioned pSB1T3 were already exist. Digestion mix double digestion(EcoRI and PstI)

Control 1(EcoRI only)

Control 2(PstI only)

[[image:|thumb|Digestion results]]

Ligation

Electrophoresis and Gel extracted

mini-prep result

Gel extraction for Ag43-dT on pSB1T3. We cut low bp band (see image below). Gel Extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics). Got 50ul of DNA solution.

Digestion

Digestion Ag43-dT on pSB1T3 and pT7-RBS on pSB1C3 to confirm ligation was succeeded or not. Ag43-dT on pSB1T3(30ng/ul) Control 1(EcoRI only)

Control 2(PstI only)

double digestion(EcoRI & PstI)

pT7-RBS on pSB1C3(40ng/ul)

Control 1(EcoRI only)

Control 2(PstI only)

double digestion(EcoRI & PstI)