Alice:LabNotes/2010-2-19: Difference between revisions

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#mix well by pipetting and the hybridization mix should be around 27-29ul
#mix well by pipetting and the hybridization mix should be around 27-29ul
#incubate the hybridization mixture at 65C for 24-72 hrs with heated lid at 105C (make sure no extensive evaporation if incubating more than 24hr)
#incubate the hybridization mixture at 65C for 24-72 hrs with heated lid at 105C (make sure no extensive evaporation if incubating more than 24hr)
==Prepare magnetic beads==
#prewarm SureSelect Wash Buffer #2 at 65C for use in next step "Select hybrid capture with SureSelect"
#resuspend and vortex the Dynal magnetic beads (Dynabeads M-280 Strapatavidin, invitrogen)
#for each hybridization, add 50ul dynalbead to 1.5ml tube
#wash the beads:
##add 200ul of SureSelect Binding Buffer
##mix the beads on a vortex for 5 sec
##put the tubes into a magnetic separator, remove and discard the supernatant
##repeat the above steps (4.1-4.3) for a total of 3 washes
#resuspend the beads in 200ul of SureSelect Binding buffer
==Select Hybrid capture with SureSelect==
#estimate the volume of hybridization mix that remained after 24-72hr incubation
#add hybridization mix directly from the thermal cycler to the bead solution, and invert the tube to mix 3-5 times
#incubate the hybrid-capture/bead solution on a nutator or shaker at RT for 30min (make sure the sample is properly mixed)
#briefly spin in a centrifuge
#separate the beads and buffer on a magnetic separator and remove the supernatant
#Resuspend the beads in 500ul of SureSelect Wash buffer #1 by mixing on a vortex for 5 sec
#incubate the samples for 15 min at RT
#wash the beads:
##separate the beads and buffer on magnetic separator and remove the supernatant
##resuspend the beads in 500ul of prewarmed SureSelect Wash Buffer #2 and mix on vortex for 5 sec
##incubate the samples for 10min at 65C
##invert the tube to mix as the beads may be settled
##repeat the above steps (8.1-8.4) for a total of 3 washes, and make sure all the wash buffer has been removed
#mix the beads in 50ul of SureSelect Elution buffer on a vortex mixer for 5 sec to resuspend the beads
#incubate the samples for 10 min at RT
#separate the beads and buffer on Dynal magnetic separator
#transfer the supernatant to a new 1.5ml tube and add 50ul SureSelect Neutralization buffer to captured DNA
#Desalt the capture solution with Qiagen Minelute column and elute in 15ul EB
==post-hybridization amplification and purification==
*Prepare the Post-Capture PCR Master Mix in a 1.5ml tube.
*Aliquot 5μl of bead-bound captured DNA as template into 3 PCR tubes/wells. Mix well by pipetting up and down 5 times.
                                                          x3
post-capture DNA:                              5ul
Phusion High-Fidelity PCR Master Mix (2x):    50μl
PCR grade water:                              41ul
Syb_FP5, 100 μM (Final Conc.: 2μM):            2ul
Syb_RP7, 100 μM (Final Conc.: 2μM):            2ul
SYBR green 50x:                              0.4ul
Total :                                        100ul
98C 30sec -> (98C 10sec -> 60C 30sec -> 72C 30sec) x 13 cycles -> 72C 5min -> hold at 4C
*Qiaquick column purification and elute in 40ul EB


=Biotin-labeled probe capture continued=
=Biotin-labeled probe capture continued=
*previous steps can be found under labnote 2/14/10
*previous steps can be found under labnote 2/15/10


*use only the foreskin set to do the following step first to find the ideal condition  
*use only the DF and foreskin sets to do the following steps to find the ideal condition  
==Prepare the Streptavidin Dynabeads==
==Prepare the Streptavidin Dynalbeads==
a. Take 50ul M-280 streptavidin Dynabeads per reaction, place the tube on magnet and remove the liquid when the solution becomes clear
#Take 50ul M-280 streptavidin Dynalbeads per reaction, place the tube on magnet and remove the liquid when the solution becomes clear
b. add twice the volume of the beads of 1x binding buffer, place the tube on magnet and remove the liquid when the solution becomes clear
#add twice the volume of the beads of 1x binding buffer, vortex for 5 sec, place the tube on magnet and remove the liquid when the solution becomes clear
c. wash the beads for a total of  3 times with the 1x binding buffer (1M NaCl, 10mM Tris-HCl pH 7.5, 1mM EDTA)
#wash the beads for a total of  3 times with the 1x binding buffer (1M NaCl, 10mM Tris-HCl pH 7.5, 1mM EDTA)
d. resuspend the beads in 200ul 1x binding buffer, warm up to 45C.  
#resuspend the beads in 200ul 1x binding buffer, warm up to 45C.  
c. Add the 20ul hybridization mix to 200ul M-280 beads, incubate at 45C in the Thermal Mixer at 300rpm for 30 min.  
#Add the 20ul hybridization mix to 200ul M-280 beads, incubate at 45C in the Thermal Mixer at 300rpm for 30 min.  
d. Remove the liquid from the beads with a magnet. Take out 1/3 of the product to go on without washing.
#Remove the liquid from the beads with a magnet. Take out 1/2 of the DF set to go on without washing.
e. Perform three 10-min wash with 0.5ml pre-warm 0.1x SSC and 0.1% SDS at 55C, 37C (take 1/3 for each temp) using Thermal Mixer.
 
f. After the final wash, resuspend the beads with 50ul 0.1M NaOH, incubate at RT for 10min.
==post-hybridization washing==
g. Separate the supernatant from the beads with a magnet, and transfer the supernatant (eluted DNA) to 70ul 1M Tris-HCl, pH 7.5.  
#Perform three 10-min wash with 0.5ml pre-warm 0.1x SSC and 0.1% SDS at 47C (1/2 DF set), 55C, 37C (take 1/2 foreskin set for these temp) using Thermal Mixer.
h. Purifythe 120ul neutralized DNA with a Qiaquick column, eluted with 30ul EB.
#After the final wash, resuspend the beads with 50ul 0.1M NaOH, incubate at RT for 10min.
#Separate the supernatant from the beads with a magnet, and transfer the supernatant (eluted DNA) to 70ul 1M Tris-HCl, pH 7.5.  
#Purify the 120ul neutralized DNA with a Qiaquick column, eluted with 30ul EB.


==Post-capture PCR==
==Post-capture PCR==
a.Set up a 400ul reaction with Phusion High-Fidelity PCR master mix:
  reaction setup:
   2x Phusion master mix:    200ul
                                        x2        x4
   100uM PCR_F       2ul
   2x Phusion master mix:    50ul
   100uM PCR_R       2ul
   100uM Sol_up            0.5ul
   50X SYBG I     3.2ul
   100uM Sol_lo            0.5ul
   Captured DNA       30ul
   50X SYBG I     0.4ul
   H2O     163ul
   Captured DNA             15ul
b.Perform real-time PCR with 98C 30s -> 20 x (98C 10s -> 63C 20s-> 72C 20s) -> 72C 2min.  
   H2O             34ul
  Terminate the reaction when the amplification curves approach to the plateau.  
  Perform real-time PCR with 98C 30s -> 20 x (98C 10s -> 63C 20s-> 72C 20s) -> 72C 2min.
 
  14 cycles for DF set without any washing involved
c.Perform PAGE size selection on 250-350bp amplicons for QPCR quantification and Illumina sequencing.
*Terminate the reaction when the amplification curves approach to the plateau.  
*PAGE gel image result:
[[File:ZhangLab_2 2010-02-24 11hr 16min.jpg]]
*measure enrichment level using QPCR (following the procotol from Nimblgen/Roche)

Latest revision as of 21:26, 5 April 2010

Agilent SureSelect Human Allexon capture[edit]

Overview and time requirements[edit]

File:Agilent sureselect protocol.jpg

Step Time
gDNA library production 1 day
library hybridization 24-72 hrs
bead preparation 30 minutes
capture selection and washing 2 hours
DNA purification 30 minutes
Post-hybridization amplification 1 hour
PCR purification 30 minutes
size selection and purification 4 hours

gDNA sample preparation[edit]

  • Agilent protocol uses Preparing Samples for Paired-End Sequencing (Illumina), our protocol uses NEBNext DNA sample Prep Master Mix Set 1 (NEB, E6040S/L)
  1. End-Repair reaction (blunt end sample with 3' phos.)
  2. Qiagen Qiaquick column purification
  3. Add ‘A’ Bases to the 3' end of the DNA fragments
  4. Qiagen Qiaquick column purification
  5. ligate the single-end adapters
  6. Qiagen Qiaquick column purification (Ampure DNA purification kit can also be used)
  7. amplify adapter-ligated samples
  8. Qiagen Qiaquick column purification and PAGE gel verification
  • previous preparation steps can be found under labnotes 1-10-2010 for DF and foreskin sets
  • previous preparation steps can be found under labnotes 1-15-2010 for CViB and CViF sets
  • DF and foreskin sets still have purified post-PCR product after ligation
  • ligation step and beyond for CViB and CViF sets can be found under labnotes 2-15-10

library hybridization[edit]

  1. Aliquot 500ng of preped gDNA libraries and concentrate at <45C down to 3.4ul. If the samples dried up completely, resuspend in 3.4ul ddH2O.
  2. mix the component according to below at RT to prepare hybridization buffer (if precipitate forms, warm the hybridization buffer at 65C for 5 minutes):
    1. For every reaction, add 25ul of SureSelect Hyb #1, 1ul of SureSelect Hyb #2 (red cap), 10ul of SureSelect Hyb #3 (yellow cap), 13ul of SureSelect Hyb #4, total is 49ul
    2. For every 4 reactions, add 100ul of SureSelect Hyb #1, 4ul of SureSelect Hyb #2 (red cap), 40ul of SureSelect Hyb #3 (yellow cap), 52ul of SureSelect Hyb #4, total is 196ul
  3. prepare the SureSelect Oligo Capture Library Mix for target enrichment (keep everything on ice for next few steps):
    1. add 5ul of SureSelect Oligo Capture library and 1ul ddH2O
    2. prepare 1:1 dilution of the RNase Block (purple cap)
    3. add 1ul of diluted RNase Block to each capture library and mix thoroughly by pipetting
    4. keep tubes on ice
  4. prepare library for target enrichment in a separate tube (for the following steps, make sure evaporation is no more than 3-4ul):
    1. put 3.4ul of prepped library(500ng) into tube B add 2.5ul of SureSelect Block #1, 2.5ul of SureSelect Block #2, and 0.6ul of SureSelect Block #3
    2. mix well by pipetting and cap the tubes
    3. heat up the prepped libraries + blockers at 95C 5min and 65C hold
    4. maintain the tubes at 65C while load 40ul of hybridization buffer into tube A (# of tubes needed is the # of prepped libraries)
    5. incubate both tubes at 65C for at least 5 min before go on
  5. put the SureSelect Oligo Capture Library from step 3.4 into tube C and incubate at 2 min (now the thermocycler should have three tubes per sample incubating at 65C)
  6. while maintaining the tubes at 65C, transfer 13ul from tube A (hybridization buffer) into tube C (SureSelect capture library) for each sample
  7. while maintaining the tubes at 65C, transfer the entire content from tube B (prepped libraries) into tube C (SureSelect capture library) for each sample
  8. mix well by pipetting and the hybridization mix should be around 27-29ul
  9. incubate the hybridization mixture at 65C for 24-72 hrs with heated lid at 105C (make sure no extensive evaporation if incubating more than 24hr)

Prepare magnetic beads[edit]

  1. prewarm SureSelect Wash Buffer #2 at 65C for use in next step "Select hybrid capture with SureSelect"
  2. resuspend and vortex the Dynal magnetic beads (Dynabeads M-280 Strapatavidin, invitrogen)
  3. for each hybridization, add 50ul dynalbead to 1.5ml tube
  4. wash the beads:
    1. add 200ul of SureSelect Binding Buffer
    2. mix the beads on a vortex for 5 sec
    3. put the tubes into a magnetic separator, remove and discard the supernatant
    4. repeat the above steps (4.1-4.3) for a total of 3 washes
  5. resuspend the beads in 200ul of SureSelect Binding buffer

Select Hybrid capture with SureSelect[edit]

  1. estimate the volume of hybridization mix that remained after 24-72hr incubation
  2. add hybridization mix directly from the thermal cycler to the bead solution, and invert the tube to mix 3-5 times
  3. incubate the hybrid-capture/bead solution on a nutator or shaker at RT for 30min (make sure the sample is properly mixed)
  4. briefly spin in a centrifuge
  5. separate the beads and buffer on a magnetic separator and remove the supernatant
  6. Resuspend the beads in 500ul of SureSelect Wash buffer #1 by mixing on a vortex for 5 sec
  7. incubate the samples for 15 min at RT
  8. wash the beads:
    1. separate the beads and buffer on magnetic separator and remove the supernatant
    2. resuspend the beads in 500ul of prewarmed SureSelect Wash Buffer #2 and mix on vortex for 5 sec
    3. incubate the samples for 10min at 65C
    4. invert the tube to mix as the beads may be settled
    5. repeat the above steps (8.1-8.4) for a total of 3 washes, and make sure all the wash buffer has been removed
  9. mix the beads in 50ul of SureSelect Elution buffer on a vortex mixer for 5 sec to resuspend the beads
  10. incubate the samples for 10 min at RT
  11. separate the beads and buffer on Dynal magnetic separator
  12. transfer the supernatant to a new 1.5ml tube and add 50ul SureSelect Neutralization buffer to captured DNA
  13. Desalt the capture solution with Qiagen Minelute column and elute in 15ul EB

post-hybridization amplification and purification[edit]

  • Prepare the Post-Capture PCR Master Mix in a 1.5ml tube.
  • Aliquot 5μl of bead-bound captured DNA as template into 3 PCR tubes/wells. Mix well by pipetting up and down 5 times.
                                                         x3
post-capture DNA:                               5ul
Phusion High-Fidelity PCR Master Mix (2x):     50μl
PCR grade water:                               41ul 
Syb_FP5, 100 μM (Final Conc.: 2μM):             2ul
Syb_RP7, 100 μM (Final Conc.: 2μM):             2ul
SYBR green 50x:                               0.4ul
Total :                                        100ul
98C 30sec -> (98C 10sec -> 60C 30sec -> 72C 30sec) x 13 cycles -> 72C 5min -> hold at 4C
  • Qiaquick column purification and elute in 40ul EB

Biotin-labeled probe capture continued[edit]

  • previous steps can be found under labnote 2/15/10
  • use only the DF and foreskin sets to do the following steps to find the ideal condition

Prepare the Streptavidin Dynalbeads[edit]

  1. Take 50ul M-280 streptavidin Dynalbeads per reaction, place the tube on magnet and remove the liquid when the solution becomes clear
  2. add twice the volume of the beads of 1x binding buffer, vortex for 5 sec, place the tube on magnet and remove the liquid when the solution becomes clear
  3. wash the beads for a total of 3 times with the 1x binding buffer (1M NaCl, 10mM Tris-HCl pH 7.5, 1mM EDTA)
  4. resuspend the beads in 200ul 1x binding buffer, warm up to 45C.
  5. Add the 20ul hybridization mix to 200ul M-280 beads, incubate at 45C in the Thermal Mixer at 300rpm for 30 min.
  6. Remove the liquid from the beads with a magnet. Take out 1/2 of the DF set to go on without washing.

post-hybridization washing[edit]

  1. Perform three 10-min wash with 0.5ml pre-warm 0.1x SSC and 0.1% SDS at 47C (1/2 DF set), 55C, 37C (take 1/2 foreskin set for these temp) using Thermal Mixer.
  2. After the final wash, resuspend the beads with 50ul 0.1M NaOH, incubate at RT for 10min.
  3. Separate the supernatant from the beads with a magnet, and transfer the supernatant (eluted DNA) to 70ul 1M Tris-HCl, pH 7.5.
  4. Purify the 120ul neutralized DNA with a Qiaquick column, eluted with 30ul EB.

Post-capture PCR[edit]

 reaction setup:
                                       x2        x4
  2x Phusion master mix:    50ul
  100uM Sol_up             0.5ul
  100uM Sol_lo             0.5ul
  50X SYBG I		    0.4ul
  Captured DNA	             15ul
  H2O 		             34ul
  Perform real-time PCR with 98C 30s -> 20 x (98C 10s -> 63C 20s-> 72C 20s) -> 72C 2min.
14 cycles for DF set without any washing involved 
  • Terminate the reaction when the amplification curves approach to the plateau.
  • PAGE gel image result:

File:ZhangLab 2 2010-02-24 11hr 16min.jpg

  • measure enrichment level using QPCR (following the procotol from Nimblgen/Roche)