Paul.xu: Created page with "= Isolation of genomic DNA from zebrafish larvae for sequencing libraries = == Preparing Embryos or Larvae == #Separate embryos or larvae (I like 100 d5) per 50mL conical t..."

2012-03-26T22:50:09Z

Created page with "= Isolation of genomic DNA from zebrafish larvae for sequencing libraries = == Preparing Embryos or Larvae == #Separate embryos or larvae (I like 100 d5) per 50mL conical t..."

New page

= Isolation of genomic DNA from zebrafish larvae for sequencing libraries =

== 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 %
| 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" |  
| 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 >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.

== DNA fragmentation using the Covaris ==

#Shear for 200nt size using the covaris (program: 200nt_nextGen
(3 min), Biopolymers Facility).

#Transfer sample from covaris tube into a 1.5 ml low-retention
Eppi, add 1 ul of 20 mg/ ml RNase A ("Purelink", Invitrogen).

#Incubate for 1 hr @ 37C.

== MinElute PCR Reaction Cleanup kit ==

#transfer sample into 1.5 ml low-bind eppendorf tube
#add 500 ul of buffer PB to sample
#Place MinElute column in and add sample to column
#Wait 2 minutes, spin @ 13,000 rpm, discard the flow-through
#Add 750 ul of buffer PE to column
#Wait 1 minutes, spin @ 13,000 rpm for 2 min, discard the
flow-through

#Spin again @ 13,000 rpm for 2 min, discard the flow-through
#place column in clean 1.5 ml Eppi
#Open column and air dry for 2 min
#Add 15 ul of buffer EB to column, let sit for 2 min, spin for 60
sec

#Add 15 ul of buffer EB to column, let sit for 2 min, spin for 60
sec

== Perform End Repair ==

#Prepare reaction mix:

{| cellspacing="1" cellpadding="1" border="0"
|-
| bgcolor="#eaeaea" | 30 ul
| bgcolor="#eaeaea" | DNA sample
|-
| bgcolor="#ffffff" | 45 ul
| bgcolor="#ffffff" | nuclease-free water
|-
| bgcolor="#eaeaea" | 10 ul
| bgcolor="#eaeaea" | T4 DNA Ligase buffer w/ 10mM ATP
(Phosphorylation Reaction Buffer)

|-
| bgcolor="#ffffff" | 4 ul
| bgcolor="#ffffff" | dNTP Mix
|-
| bgcolor="#eaeaea" | 5 ul
| bgcolor="#eaeaea" | T4 DNA Polymerase
|-
| bgcolor="#ffffff" | 1 ul
| bgcolor="#ffffff" | Klenow DNA Polymerase, Large Fragment
|-
| bgcolor="#eaeaea" | 5 ul
| bgcolor="#eaeaea" | PNK T4
|-
| bgcolor="#ffffff" | 100 ul
| bgcolor="#ffffff" | final vol.
|}

#Incubate in thermal cycler for 30 min at 20C

== MinElute PCR Reaction Cleanup kit ==

#transfer sample into 1.5 ml low-bind eppendorf tube
#add 500 ul of buffer PB to sample
#Place MinElute column in and add sample to column
#Wait 1 minutes, spin @ 13,000 rpm, discard the flow-through
#Add 750 ul of buffer PE to column
#Wait 1 minutes, spin @ 13,000 rpm for 1 min, discard the
flow-through

#Spin again @ 13,000 rpm for 1 min, discard the flow-through
#place column in clean 1.5 ml Eppi
#Add 32 ul of buffer EB to column, let sit for 1 min, spin for 1
min @ 13,000 rpm

== Add "A" Bases to Fragments ==

#Prepare reaction mix:

{| cellspacing="1" cellpadding="1" border="0"
|-
| bgcolor="#eaeaea" | 32 ul
| bgcolor="#eaeaea" | DNA sample
|-
| bgcolor="#ffffff" | 5 ul
| bgcolor="#ffffff" | Klenow Buffer (NEBuffer 2)
|-
| bgcolor="#eaeaea" | 10 ul
| bgcolor="#eaeaea" | 1 mM ATP
|-
| bgcolor="#ffffff" | 3 ul
| bgcolor="#ffffff" | Klenow Fragment, Exo-
|}

#Incubate 30 minutes @ 37C

== MinElute PCR Reaction Cleanup kit ==

#transfer sample into 1.5 ml low-bind eppendorf tube
#add 250 ul of buffer PB to sample
#Place MinElute column in and add sample to column
#Wait 1 minutes, spin @ 13,000 rpm, discard the flow-through
#Add 750 ul of buffer PE to column
#Wait 1 minutes, spin @ 13,000 rpm for 1 min, discard the
flow-through

#Spin again @ 13,000 rpm for 1 min, discard the flow-through
#place column in clean 1.5 ml Eppi
#Add 10 ul of buffer EB to column, let sit for 1 min, spin for 1
min @ 13,000 rpm

== Ligate Adaptors to Fragments and Purify ==

#Prepare reaction mix:

{| cellspacing="1" cellpadding="1" border="0"
|-
| bgcolor="#eaeaea" | 10 ul
| bgcolor="#eaeaea" | DNA sample
|-
| bgcolor="#ffffff" | 25 ul
| bgcolor="#ffffff" | 2x DNA Ligase Buffer (Quick Ligation
Reaction Buffer)

|-
| bgcolor="#eaeaea" | 10 ul
| bgcolor="#eaeaea" | Adapter Oligo (maintain a 10:1
adapter/insert ratio - 5ug/10ul)

|-
| bgcolor="#ffffff" | 5 ul
| bgcolor="#ffffff" | DNA Ligase (Quick T4 DNA Ligase)
|}

#Incubate at RT for 15 minutes

== MinElute PCR Reaction Cleanup kit ==

#transfer sample into 1.5 ml low-bind eppendorf tube
#add 250 ul of buffer PB to sample
#Place MinElute column in and add sample to column
#Wait 1 minutes, spin @ 13,000 rpm, discard the flow-through
#Add 750 ul of buffer PE to column
#Wait 1 minutes, spin @ 13,000 rpm for 1 min, discard the
flow-through

#Spin again @ 13,000 rpm for 1 min, discard the flow-through
#place column in clean 1.5 ml Eppi
#Add 32 ul of buffer EB to column, let sit for 1 min, spin for 1
min @ 13,000 rpm

== Size Selection on 1-1.5% Agarose Gel and QuiaQuick Gel extraction ==

#Prepare 1-1.5% Agarose gel
#Load Sample and run for 30 min @ >150V
#Cut out band (~266 bp) and weigh gel slice
#Add 3 gel volumes of Buffer QG and melt @50C for 10 min
#Add 1 gel volume of isopropanol (add 1-2 ul of 3M NaAc, pH5.5 if
buffer is orange to purple)

#Place MinElute column in and add sample to column
#Wait 2 minutes, spin @ 13,000 rpm, discard the flow-through
#Add 750 ul of buffer PE to column
#Wait 1 minutes, spin @ 13,000 rpm for 1 min, discard the
flow-through

#Spin again @ 13,000 rpm for 1 min, discard the flow-through
#place column in clean 1.5 ml Eppi
#Add 10 ul of buffer EB to column, let sit for 1 min, spin for 1
min @ 13,000 rpm

== Enrich the Adaptor-Modified DNA Fragments by PCR ==

#Prepare reaction mix:

{| cellspacing="1" cellpadding="1" border="0"
|-
| bgcolor="#eaeaea" | 1 ul
| bgcolor="#eaeaea" | DNA sample
|-
| bgcolor="#ffffff" | 25 ul
| bgcolor="#ffffff" | 2x Flash Phusion Master Mix (Phusion High
Fidelity PCR Master Mix, Finnzymes)

|-
| bgcolor="#eaeaea" | 1 ul
| bgcolor="#eaeaea" | PCR primer 1.1
|-
| bgcolor="#ffffff" | 1 ul
| bgcolor="#ffffff" | PCR primer 2.1
|-
| bgcolor="#eaeaea" | 22 ul
| bgcolor="#eaeaea" | Nuclease-free water
|}

#Run the following PCR program:

{| cellspacing="1" cellpadding="1" border="0"
|-
| bgcolor="#eaeaea" | Cycles
| bgcolor="#eaeaea" | Duration
| bgcolor="#eaeaea" | Temperature
|-
| bgcolor="#ffffff" | 1
| bgcolor="#ffffff" | 30 sec
| bgcolor="#ffffff" | 98C
|-
| bgcolor="#eaeaea" | 10
| bgcolor="#eaeaea" |  
| bgcolor="#eaeaea" |  
|-
| bgcolor="#ffffff" | -
| bgcolor="#ffffff" | 10 sec
| bgcolor="#ffffff" | 98C
|-
| bgcolor="#eaeaea" | -
| bgcolor="#eaeaea" | 30 sec
| bgcolor="#eaeaea" | 65C
|-
| bgcolor="#ffffff" | -
| bgcolor="#ffffff" | 30 sec
| bgcolor="#ffffff" | 72C
|-
| bgcolor="#eaeaea" | 1
| bgcolor="#eaeaea" | 5 min
| bgcolor="#eaeaea" | 72C
|-
| bgcolor="#ffffff" | 1
| bgcolor="#ffffff" | hold
| bgcolor="#ffffff" | 4C
|}

== Size Selection on 1-1.5% Agarose Gel and QuiaQuick Gel extraction (optional: can be purified on E-gel by Biopolymers Facility) ==

#Prepare 1-1.5% Agarose gel
#Load Sample and run for 30 min @ >150V
#Cut out band (~266 bp) and weigh gel slice
#Add 3 gel volumes of Buffer QG and melt @50C for 10 min
#Add 1 gel volume of isopropanol (add 1-2 ul of 3M NaAc, pH5.5 if
buffer is orange to purple)

#Place MinElute column in and add sample to column
#Wait 2 minutes, spin @ 13,000 rpm, discard the flow-through
#Add 750 ul of buffer PE to column
#Wait 1 minutes, spin @ 13,000 rpm for 1 min, discard the
flow-through

#Spin again @ 13,000 rpm for 1 min, discard the flow-through
#place column in clean 1.5 ml Eppi
#Add 30 ul of buffer EB to column, let sit for 1 min, spin for 1
min @ 13,000 rpm

#Submit library for quality control and sequencing

[[Category:Protocols]]

Library Prep for NextGen Sequencing - Revision history

Paul.xu: Created page with "= Isolation of genomic DNA from zebrafish larvae for sequencing libraries = == Preparing Embryos or Larvae == #Separate embryos or larvae (I like 100 d5) per 50mL conical t..."