Genotyping

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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 for each sample.
• Centrifuge the tubes after this step

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.

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PCR

1. GSK 3-Alpha Flox

a) Primers :

• GSK3 alpha flox Forw 5’ccc cca cca agt gat ttc act gct a
• GSK3 alpha flox Rev 5’ ctt gaa cct ttt gtc ctg aag aac c

b) PCR conditons :

• 1)94 deg for 5 mins
• 2)94 deg for 30sec
• 3)55 deg for 30sec
• 4)68 deg for 1.25 min

Step 2-4 for 35 cycles

• 5) 68 deg for 7 mins

c) Proportions in microliters : PCR Reaction Mix– 10 (Sigma Red Mix)

Primers	Volumes (uL)
PCR Reaction Mix	10
Primer 1	1
Primer 2	1
Water	4
DNA	4
TOTAL	20

d) Notes : ~ 500bp= wtband/ ~ 650bp = floxed band

2) GSK 3-Beta Flox

a) Primers :
Forward Primer : GGG GCA ACC TTA ATT TCA TT
Reverse Primer : TCT GGG CTA TAG CTA TCT AGT AAC G

b) PCR conditons :

• 1) 94 deg for 4 mins
• 2) 94 deg for 30sec
• 3) 54 deg for 30sec
• 4) 72 deg for 2 min

Step 2-4 for 36 cycles

• 5) 72 deg for 7 mins

c) Proportions in microliters : PCR Reaction Mix – 10 (Sigma Red Mix)

Primers	Volumes (uL)
PCR Reaction Mix	10
Primer 1	1
Primer 2	1
Water	4
DNA	4
TOTAL	20

d) Notes : ~ 895bp= wtband/ ~ 1100bp = floxed band

CRE Multiplex with internal control (for MerCreMer)

a) Primers

• JAX49: 5'- TCCAATTTACTGACCGTACACCAA-3
  
• JAX50: 5'- CCTGATCCTGGCAATTTCGGCTA-3
  
• JAX38: 5'- CTAGGCCACAGAATTGAAAGATCT-3
  
• JAX39: 5'- GTAGGTGGAAATTCTAGCATCATCC-3
  

b) Proportions in microliters : PCR Reaction Mix – 10 (Sigma Red Mix)

Primers	Volumes (uL)
PCR Reaction Mix	10
Primers	Primers 1 uL each
Water	2
DNA	4
TOTAL	20

d) Cycling

Steps #	Temp (C)	Time	Note
1	95	3 min	---
2	95	30 sec	---
3	62	30 sec	---
4	72	1:00	---
5	---	---	Go to step 2-4, 35 times
6	72	5 min	---
7	10	---	hold

c) Notes : ~ 500bp = MerCreMer Band/ ~ 300bp = internal control