Genotyping Mouse Tail Tips

(CLEAN METHOD- for difficult PCR reactions or for gels that will go into papers)

 

Tail lysis buffer

0.2% SDS

0.1M Tris pH8.5

5mM EDTA

200mM NaCl

 

Proteinase K: 10mg/mL stock solution (Sigma P8044)

TE Buffer (optional): 10mM Tris pH8-8.5 and EDTA 1mM

 

   1. Add 600 uL tail lysis buffer with 30 uL proteinase K to tail tip.

   2. Keep agitated in 55íC shaking water bath overnight.

   3. Centrifuge at 15000 x g for 20 minutes at 4íC.

   4. Take 400 uL of supernatant (avoid floating protein) and transfer to new Eppendorf tube, being careful to avoid dislodging pellet.

   5. Add 600 uL of isopropanol and gently invert to mix.

   6. Leave overnight at 4íC (or a few hours).

   7. Perform high speed centrifugation (15000 x g for 20 minutes) to spin down DNA.

   8. Gently pour off isopropanol to avoid dislodging pellet (some isopropanol can remain).

   9. Add 400 uL of 70% ethanol gently along the wall of the tube.

  10. Perform high speed centrifugation (15000 x g for 10-15 minutes) to spin down DNA.

  11. Pour off ethanol and allow to dry inverted for 30 minutes.

  12. Add 100 uL of water (or TE buffer) and let sit overnight at room temperature (or a few hours on the heat block).

  13. DNA is ready for PCR.

 

 Tail Digestion: Hot Shot Genomic DNA Preparation

(DIRTY METHOD- GOOD FOR MOST APPLICATIONS)

Protocol attained from Tenan Lab, Pu Zhang, Modified by Leah Contrino 06/2006

 

DNA preparation from mouse tail for PCR genotyping:

1. Obtain 1-2mm sample and place into a 1.5mL eppendorf tube.

2. Add 100uL 50mM NaOH to the same tube and heat to 96íC for 1 hour (or  until dissolved), vortexing every 15-20 minutes.

3. Vortex samples once more and add 25uL 1M tris-HCl, pH 6.8 to neutralize the NaOH and spin down at 13k x g for 2 minutes.

4. Transfer supernatant to new tube. Use 1-2uL in a 20μL PCR reaction.

Additional notes:

- The protocol is adapted from the protocol of the book Manipulating Mouse Embryo, 3rd addition, pg 529-530.

- 50mM NaOH: 0.1g in 50mL

 

PCR

 

PCR recipe (each sample):

DNA                                                                           1-2 μL

EconoTaq PLUS GREEN 2X Master Mix       10 μL

Primers (10μM)                                                    1 μL each (forward and reverse)

Water                                                                        X μL

Total:   20 μL

           

OR:

 

DNA                                                                                       1-2 μL

10X PCR Buffer                                                                 2 μL

5mM dNTPs                                                                       2 μL

Primers (10μM)                                                                1 μL each (forward and reverse)

Taq Polymerase (Denville blue)                                 0.1 μL

Water                                                                                   X μL

                                                                        Total:            20 μL

 

 

Make enough master mix (everything but DNA) for 1.2x the number of samples you have.