Megabase DNA Extraction from Animal Blood
Collect 3mL animal's blood in 5 mL EDTA anticoagulative tube , and store at 4 °C .
Mix blood by gently rocking for 10min at room temperature to ensure a homogenous WBC distribution .
Transfer 3ml blood to 15ml screw cap tube containing 9ml RBC lysis solution ( 3 volumes ) , and mix by gently inverting 10x .
Incubate 5 min at room temperature .
Gently mix by inverting at least once during incubation .
Spin 5 min at 2000xg at 4⁰C and carefully pipet out supernatant while not disturbing the pellet .
Resuspend cell pellet in 3ml PBS ( 1 blood volume ) :
Add 9ml rbc lysis sol ( 3 volumes ) , invert to mix and incubate 5 min at room temperature .
Spin cell suspension for 5 min at 2000xg at 4⁰C and carefully pipet out supernatant while not disturbing the pellet .
Remove the last drop of liquid by tilting the tube and removing liquid with pipet tip .
Resuspend cell pellet in 580ul cell suspension buffer ( Bio Rad plug lysis kit ) until homogenous suspension .
Transfer 375ul to a new microfuge tube and label ( 400 ) .
Transfer 187.5ul to another tube labeled ( 200 ) ; add 187.5ul cell suspension buffer , and pipet mix gently 5x .
Keep both tubes on ice until ready to embed in agarose ( section II ) .
Melt 2 % agarose ( Bio Rad kit ) by immersing in microwave - boiled water for 10 - 15min until agarose is completely melted .
Equilibrate both tubes from step 11 section I in 43°C water bath / heat block for at least 5 min before use .
After 5 min , prepare 5 plugs from one tube by performing the next steps rapidly to avoid solidification of the cell - agarose mixture before pipeting into plug mold :
Place plug cast on ice for at least 45min or until agarose has solidified .
Alternatively , can place plug cast on inverted metal microfuge ice block on ice for 45min , to avoid potential contact of agarose with ice .
Wash away hemoglobin and cellular material with Proteinase K and Wash Buffer .
