DNA Preparation

DNA Preparation

Tissue collection The method of tissue collection must be described in the investigator’s Animal Use Protocol and approved by Loyola University Chicago’s IACUC. Tissues that can be used as a source of genomic DNA include:

• Tail clip
• Ear punch

Genotyping is recommended prior to weaning so appropriate cage cards can be generated and so that the animal numbers are deducted from the appropriate strain and pain category on the approved protocol

HOTSHOT method (G.E. Truett, et al, BioTechniques 29:52-54, July 2000) Cut 1-2 mm tail and place in a 0.5 ml microfuge tube. Caution - larger pieces of tail can inhibit the PCR. Add 75 µl Alkaline Lysis Reagent. Assure that the tail fragment is completely submerged. Incubate at 95C for at least one hour (longer may be better – see below) and then store at 4C until you proceed to the next step. A thermocycler is convenient for this step. Add 75 µl Neutralization Reagent using a new aerosol-barrier tip for each sample. Mix well, using tip to break up tissue. Some people like to centrifuge the tubes after this step and transfer the neutralized supernatant to a new tube, but this is not necessary. When genotyping animals that are 6 weeks and older, we find that increasing the 95C incubation time to 2 hr yields better results. Preps made from tail pieces longer than 2mm may inhibit the PCR. Use 1 µl of neutralized supernatant per 20 µl PCR reaction.

Alkaline Lysis Reagent to 25 ml water, add 62.5 µl of 10 N NaOH (final conc is 25 mM) 10.0 µl of 0.5 M disodium EDTA (final conc is 0.2 mM) (pH should be about 12, but should not have to be adjusted) Make fresh every 1-2 months. Keep solution at RT. Note: because of the presence of EDTA in the alkaline lysis reagent, you may need to increase the amount of magnesium chloride in your PCR master mix. Your PCR conditions may need to be re-optimized when switching to this method of DNA preparation. Neutralization Reagent To 24 ml water, add 1 ml of 1 M Tris-HCl (final conc is 40 mM) (pH should be about 5, but should not have to be adjusted) Keep solution at RT. Make 1 M Tris-HCl with Tris hydrochloride salt