Britt Lab PresentationpSHR::NTF

Ryan Hamstengel

Purpose: Identify Cell Death Related Transcripts

Procedure for pSHR::NTF Experiment1. PCR pSHR2. Gel Extraction3. DTopo Vector Creation4. Confirm by Sequencing6. LR Clonase Reaction (B-Intact Creation)6. Grow E.Coli7. Extract E.Coli from plates8. Transform into E.Coli9. Confirm by sequencing10. Transform into Arabidopsis using Agrobacterium11. RNA Extraction

PCR pSHR

PCR Product of pSHR and pWOL

Gel Extraction: Cutting out the Bands

1. Open a disposable scalpel.2. Wear Face Shield, make sure nobody is around.

3. Turn on UV light table.4. Cut out desired bands as precisely as possible.

5. Weigh gel.

LR Clonase

Perform the Topo Cloning Reaction1. Set up the following Topo Cloning reaction. Use a 2:1 molar ratio of PCR product:Topo vector for optimal results.

2. Mix gently and incubate for 5 minutes at room temperature.3. Place on ice and proceed to transform One Shot chemically competent E.Coli.

Reagent Volume

PCR product 1 ul

Salt solution 1 ul

Water 2.5 ul

Topo vector 1.5 ul

Total Volume 6 ul

DTopo Vector

First Sequencing

Second Sequencing

Alignment of the “Short Root” promoter that was successfully cloned into the D-topo vector. We overlapped the short root promoter DNA with vector DNA to see if it had a short root promoter in it. We created primers that annealed to the original vector DNA, which then amplified the region that revealed short root promoter DNA that recombined into it. It also showed that no mutations occurred in the promoter or vector region.

Grow E.Coli1. Pipette .1ml of inoculum

volume onto plate.2. Flame sterilize spreader with

70% isopropyl or ethanol.3. Place spreader on plate and

apply even circular movements.4. Avoid disturbing plates for 10

to 20 minutes after spreading.5. After the spread plates have

been permitted to absorb the inocula for 10 to 20 minutes they may be inverted and incubated as desired.

Extracting E.Coli from Plates1. Make a 20.02ml solution of 20ml LB (“Luria Broth” nutrients) and 20ul of 1000X 50mg/ml Kanamycin since the bacteria are Kanamycin resistant.

2. Pour 8 2ml solution tubes and 1 control tube.3. Poke chosen bacteria cultures with 20ul pipette tip and then put it into chosen 2ml solution tube that is properly labelled.4. Put tubes onto “shaker.”

Transform Chemically Competent E.Coli

1. Add 2 ul of the vector into a vial of One Shot chemically competent E. Coli cells and mix gently.

2. Incubate on ice for 5 to 30 minutes.3. Heat-shock the cells for 30 seconds at 42C without shaking. Immediately transfer the tube to ice.

4. Add 250 ul of room temperature S.O.C. medium5. Spread 50-200 ul of bacterial culture on a prewarmed selective plate and incubate overnight at 37C.

Future Procedure for Extracting RNA