Editing Library Prep for NextGen Sequencing (section)

== Preparing Embryos or Larvae  ==

#Separate embryos or larvae (I like 100 d5) per 50mL conical tube 
#Remove all egg water 
#If larvae are hatched, continue with step 9. Else, to dechorionate, add ~500uL pronase to each sample) 
#Incubate 3 min. @ RT (gently shake at beginning of incubation 
for 3-4 sec.) 

#Add Ca-free Ringer's up to the 50mL mark, squirting gently along 
side of tube using plastic squeeze bottle. Add a little at a time to 
each tube instead of adding total volume to each tube at once. 
#Pour off most of Ringer's, repeat wash, being gentle and 
squirting along sides of tubes 

#Repeat wash and pour off Ringer's for a total of three washes. 
#Remove as much supernatant as possible and gently transfer to 
1.5mL tubes with transfer pipette 

#Wash once with 1X PBS, then remove as much as possible 
#Add 50 ul per larva, up to 1 ml DNA extraction buffer and 
homogenize with a 3 ml syringe (20-23G needle), at least 10 passes. 


DNA Extraction buffer (makes 10 ml) 

{| cellspacing="1" cellpadding="1" border="0"
|-
| bgcolor="#eaeaea" | final conc. 
| bgcolor="#eaeaea" | stock 
| bgcolor="#eaeaea" | volume
|-
| bgcolor="#ffffff" | 10 mM Tris pH 8.2 
| bgcolor="#ffffff" | 1M 
| bgcolor="#ffffff" | 100 ul
|-
| bgcolor="#eaeaea" | 10 mM EDTA 
| bgcolor="#eaeaea" | 0.5M 
| bgcolor="#eaeaea" | 200 ul
|-
| bgcolor="#ffffff" | 200 mM NaCl 
| bgcolor="#ffffff" | 1M 
| bgcolor="#ffffff" | 2 ml
|-
| bgcolor="#eaeaea" | 0.5% SDS 
| bgcolor="#eaeaea" | 10&nbsp;% 
| bgcolor="#eaeaea" | 500 ul
|-
| bgcolor="#ffffff" | 200 µg/ml 
proteinase K 

| bgcolor="#ffffff" | 20 mg/ml 
| bgcolor="#ffffff" | 100 ul
|-
| bgcolor="#eaeaea" | H2O 
| bgcolor="#eaeaea" | &nbsp; 
| bgcolor="#eaeaea" | 7.1 ml
|}



#Incubate @37C for 1-2 hrs (until fully dissolved), vortexing briefly every 30 min 
#Add 0.6 ml isopropanol per tube and incubate @ -20C for at least 
30 min (I like &gt;2 hrs). 

#Centrifuge @4C for 15 min, max speed. 
#Wash with 1 ml of 70% EtOH, then centrifuge again @4C for 5 min, 
max speed. 

#Remove EtOH by pipetting, spin briefly, remove remaining EtOH. 
#Resuspend in 100 ul TE. Keep on ice. Freeze or proceed to 
shearing.