Making of DNA template for riboprobe preparation
A. Plasmid DNA linearization and purification
Linearize 20ug of plasmid DNA with appropriate Enzyme:
Plasmid 20 mg
Add restrict enzyme 60 100 units ( usually 3-5 ml)
10 X buffer 10-20 ml
Add H2O, make the volume up to 100-200 ml
Incubate at 37°C (or appropriate temperature) O/N.
Check the digestion by running an agarose gel next day.
If digestion was completed, move to next step; if digestion was uncompleted, isolate the completely digested fragment by excising it from gel (moving to protocol Part B )
Extract the protein from reaction:
Make up the reactions volume to 400 500 ml
Add 400ul phenol, vortex for 20 sec.
Spin at Max speed for 5 min.
Transfer top phase into a new tube. Discard bottom phase(phenol) in appropriate way.
Add 400 ml chlorophorm to the new tube, vortex for 20 sec.
Spin at Max speed for 5 min.
Transfer the top phase into a new tube.
Repeat the chlorophorm extraction procedure as above.
Precipitate the DNA template:
Add 40 ml of 3M Sodium Acetate, pH5.5, Mix well. Add 1000 ml ice cold ETOH, Mix well.
Sit the tube in dry ice ethanol for 20 min.
Spin at top speed (~13000 rpm) for 10min. Drain the supernatant carefully.
Wash the white pellet with 70% ethanol X 2, dont over dry the pellet.
Resuspend the pellet in 40 ml DEPC H2O (Use 1mg as template for RNA probe synthesis).
B. Use PCR product as riboprobe template
Run the PCR with the right primers and make sure the product is what desired (best size during 500-800 bp).
Use QIAquick Gel Extraction kit to purify the PCR product:
Excise the DNA fragment from the agarose gel with clean, sharp scalpel.
Weigh the gel slice in a colorless tube, add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 ml).
Incubate at 500C until the gel slice has completely dissolved. Vortexing few times during the incubation.
Add 1 volume of isopropanol to the sample and mix well.
Place a QIAquick spin column in a provided 2 ml collection tube.
Apply the sample to the QIAquick column, and centrifuge for 1 min
Discard flow-through and place QIAquick column back in the same collection tube
Add 0.75ml of Buffer PE to the column and centrifuge for 1 min to wash the column
Discard the flow-through and centrifuge the column for an additional 1 min at 13,000 rpm.
Place the column into a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 50 ml of Butter EB or H2O to the center of the column membrane and centrifuge for 1 min.
Take 1 ml elution to run the appropriate agarose gel to confirm the size and purity of the template.
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