Protocol for high throughput genotyping.
Principles to speed up genotyping, and reduce human errors:
1) Reduce Sample handling by using multichannel pipettes as much as possible.
2) Pre-make and aliquot 1x mastermix
3) Do all steps on ice to avoid amplifying too much primer dimer.
Prep DNA
1) Harvest Tails from pups and put into 8 well strips or 96 well plate.
2) Add 200 uls of Tail Digestion Buffer.
3) Put strips into PCR machine with the following program:
“TailPrep”
With heated lid.
1) 50 C, 16 hours (overnight)
2) 99 C, 10 minutes
3) 4 C, 1 second.
4) End
4) Remove tubes and flick briefly. Tail should mostly disintegrate.
5) Spin down debris in tubes quickly (10 sec) either in microcentrifuge, or in plate
spinner.
Prep PCR (for 48 reactions, scale up as need be)
1) Remove premade reaction mix (in 8 well strip) from freezer and allow to thaw to 4 C
on ice or in aluminum block on ice. Each tube of strip contains 125 uls of reaction
mix, enough for 48 reactions.
2) Using multichannel pipettor, transfer 19 uls into each well of ½ of a 96 well plate, or
6 PCR strips, on ice, with filter tips.
3) Using multichannel pipettor, add 1 ul of templates to all reactions with filter tips. Be
sure to include a eGFP positive, eGFP negative and water control.
4) Apply adhesive cover, and seal very very firmly with roller(plate) or close tubes.
5) Mix wells by flicking very thoroughly.
6) Spin down quickly in plate spinner of J6M (500 RPM)) (plates) or microcentrifuge
(strips)
7) Put in machine and run the following program
“Act2-GFP” (1.5 hour run time)
With heated lid
1) 94 C for 10 minutes
2) 94 C for 30 seconds
3) 63 C for 30 seconds
4) 72 C for 30 seconds
5) Goto “2” 31 times
6) 72 C for 5 minutes
7) 4 C for 1 second
8) End
Pour and Run Gel
Mix 100 mls of 1x TAE with 2 grams of Seakem LE agarose in 250 ml erlenmyer flask
and microwave 90 seconds or until agar is dissolved.
1) Cool slightly (to 60 C)
2) Add 3 uls of 10 mg/ml EtBr.
3) Pour into a casting tray with multichannel compatible combs
4) Allow to set
5) When PCR is finished, open plate or tubes.
6) Using multichannel pipette, mix approximately 5 uls of 6x sample loading dye with
each solution, and load 5 uls of product/dye mix into gel
7) Load 1 KB plus ladder.
8) Run at 110 V for ½ an hour, or until dye bands have migrated an inch or so.
9) Take a picture. Egfp positive mice should have bands a 150 and 350. EGFP negative
mice should have bands a 150 (actin) only. If neither band is apparent, rerun the
sample – the PCR didn’t work at all. Sometimes for high copy number mice, only
EGFP will amplify (no actin). This is okay.
Recipes for Reagents:
PCR mix:
Total volume
H20
Qiagen 10x buffer(15mM
MgCl2)
dNTP (10mM)
Egfp 3' (10 uM)
Egfp 5' (10 uM)
B-Actin-FW (10 uM)
B-Actin-RV (10 uM)
Qiagen Taq
For
50
1026
777.6
For
500
10260
7776
108
21.6
27
27
27
27
10.8
1080
216
270
270
270
270
108
Make up on ice (if it gets warm, you will mostly amplify primer-dimers).
Aliquot into 8 well strips (125 uls each well). Freeze at -20. After thawing, don’t re-use.
Each strip is good for @ 48 reactions.
Primer Name
EGFP 5'
Stock
concentration
100 uM in TE
EGFP 3'
B-actin-RV
B-actin-FW
100 uM in TE
100 uM in TE
100 uM in TE
Primer sequence
CCT ACG GCG TGC AGT GCT TCA GC
CGG CGA GCT GCA CGC TGC CGT
CCT C
CAA TAG TGA TGA CCT GGC CGT
AGA GGG AAA TCG TGC GTG AC
Working dilution is 1:10 in h20 (10uM)
Notes
from Gensat protocol
from Gensat protocol
(150 bp product)
(150 bp product)
Tail Lysis Buffer recipe:
dH2O
-700ml
1M Tris(pH8.8) -50ml
0.5M EDTA - 2ml
Tween20(10%) - 50ml
Fill to 1L with dH2O
Immediately before use add 4 uls/ml of 10 mg/ml Proteinase K.