Matt:LabNotes/2014-6-18: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Mzcai
>Mzcai
mNo edit summary
Line 72: Line 72:
#*NxtraPosControl (gDNA): 0.803 ng/ul
#*NxtraPosControl (gDNA): 0.803 ng/ul
#*NxtraNegControl (H2O): 0 ng/ul
#*NxtraNegControl (H2O): 0 ng/ul
# Add 1 ul transposase (1:50 dilution) to each sample. Incubated 5.5 minutes at 55 degC
# Add 1 ul transposase (1:50 dilution) to each sample. Incubated 5 minutes at 55 degC
# Dilute Qiagen protease 1:100 in H2O. Add 1 ul to each sample and incubate 10 minutes at 50 degC, followed by 20 minutes at 70 degC
# Dilute Qiagen protease 1:100 in H2O. Add 1 ul to each sample and incubate 10 minutes at 50 degC, followed by 20 minutes at 70 degC
# Make the master mix for Exo- treatment: 1 ul Exo- and 0.4 ul dNTP per reaction
# Make the master mix for Exo- treatment: 1 ul Exo- and 0.4 ul dNTP per reaction
# Add 1.4 ul Exo- mix to each sample and incubate 15 minutes at 37 degC, followed by 20 minutes at 75 degC
# Add 1.4 ul Exo- mix to each sample and incubate 15 minutes at 37 degC, followed by 20 minutes at 75 degC
====# Finish first round PCR master mix (per reaction):
## 15 ul 2X KAPA fast SYBR.
## 0.6 ul Adapter 1.
## 1.4 ul H2O.
# Add 0.6 of barcode adapters to each sample
#*Random Primer Mix: 26
#*Oligo (dT)23 VN: 27
#*FISSEQ_RT: 28
#*Top48 Hexamer: 29
#*NTC: 30
#*No Enzyme: 31
#*NxtraPosControl (gDNA): 32
#*NxtraNegControl (H2O): 33
#*NxtraPCRControl (H2O): 34
# Add 17 ul of first round PCR master mix to each sample
#*17ul H2O for NxtraPCRControl
# Incubate in thermocycler:
## 95 degC for 1 min.
## 7 cycles:
### 95 degC for 10 sec.
### 62 degC for 15 sec.
### 72 degC for 45 sec.
# Finish second round PCR master mix:
## 35 ul KAPA fast SYBR
## 2 ul Primer 1
## 2 ul Primer 2
## 31 ul H2O
# Add 70 ul of second round PCR master mix to each sample.
# PCR program is as follows:
## 95 degC for 1 min.
## 10 cycles:
### 95 degC for 10 sec.
### 60 degC for 15 sec.
### 72 degC for 45 sec.
# Incubate in thermocycler an additional 3 min @ 72 degC for final extension.
[[]]
# Run 5 ul sample on PAGE gel with 3 ul 6X loading dye to identify positives.
## 0.5 ul low mass ladder (Invitrogen). Equalize volume with TBE.
## 25 min @ 250 V.
## Stain 5-10 min in ~100 ml 1X TBE with 6 ul SYBR Safe.
[[]]
<!--# Add 100 ul magnetic beads to each positive sample. Mix by pipetting and incubate 8 minutes off magnet.
# Place on magnet 5 minutes, then remove and discard supernatant.
# On magnet, wash 2x with 180 ul 80% EtOH. Let each wash sit 30 seconds removing.
# Let dry 5 minutes.
# Resuspend beads off magnet with 20 ul H2O and incubate 2 minutes, then return to magnet for 1 minute.
# Extract supernatant and transfer to 0.2 ml tube. Proceed directly to size selection or store at 4 degC.-->

Revision as of 23:18, 20 June 2014

Repeat Top48 Hexamer RT Primer in vitro Validation

Procedure

First Strand Synthesis

  • Mix RNA in Primer and H2O
  • Denature for 5 minutes at 70C
    • Put on ice right after
  • Add Reaction Mix and Enzyme
  • Incubate for 5 min at 25C
  • Incubate for 1 hour at 42C
  • Inactivate enzyme for 5 min at 80C
Random Primer Mix ' Oligo d(T)23 VN ' FISSEQ_RT ' Top48 Hexamer ' NTC ' No Enzyme '
Components Volume Components Volume Components Volume Components Volume Components Volume Components Volume
1.3ug/ul UHRR 0.5 1.3ug/ul UHRR 0.5 1.3ug/ul UHRR 0.5 1.3ug/ul UHRR 0.5 1.3ug/ul UHRR 0 1.3ug/ul UHRR 0.5
Random Primer Mix (60uM) 2 Oligo d(T)23 VN (50uM) 2 FISSEQ_RT (100uM) 1 Top48 Hexamer (100uM) 1 Random Primer Mix (60uM) 2 Random Primer Mix (60uM) 2
H2O 5.5 H2O 5.5 H2O 6.5 H2O 6.5 H2O 6 H2O 5.5
Denature Step Denature Step Denature Step Denature Step Denature Step Denature Step
M-MuLV Reaction Mix 10 M-MuLV Reaction Mix 10 M-MuLV Reaction Mix 10 M-MuLV Reaction Mix 10 M-MuLV Reaction Mix 10 M-MuLV Reaction Mix 10
M-MuLV Enzyme 2 M-MuLV Enzyme 2 M-MuLV Enzyme 2 M-MuLV Enzyme 2 M-MuLV Enzyme 2 H2O 2

Second Strand Synthesis

  • Add 48 ul H20
  • Add 8ul 10x Second strand synthesis reaction buffer
  • Add 4ul Second strand synthesis enzyme mix
  • Mix by pipetting
  • Incubate for 2.5 hours at 16C

Purification & Measure Concentration

  • Qiagen PCR Purification
    • Elute with 30ul
  • Nanodrop quant:
    • Random Primer Mix: 15.2 ng/ul
    • Oligo (dT)23 VN: 23.6 ng/ul
    • FISSEQ_RT: 9.7 ng/ul
    • Top48 Hexamer: 24.3 ng/ul
    • NTC: 14.5 ng/ul
    • No Enzyme: 25.0 ng/ul
  • Negative controls (NTC & No Enzyme) did not show significantly less DNA, could be RNA absorbing light and "fooling" the Nanodrop
  • Actual concentrations probably much less since previous comparison with Qubit showed 25x less

Nextera Tagmentation

  1. Add 2 ul HMW buffer to each PCR tube (8 total)
  2. Transfer 7ul of each sample to tubes
    • Random Primer Mix: 15.2 ng/ul
    • Oligo (dT)23 VN: 23.6 ng/ul
    • FISSEQ_RT: 9.7 ng/ul
    • Top48 Hexamer: 24.3 ng/ul
    • NTC: 14.5 ng/ul
    • No Enzyme: 25.0 ng/ul
    • NxtraPosControl (gDNA): 0.803 ng/ul
    • NxtraNegControl (H2O): 0 ng/ul
  3. Add 1 ul transposase (1:50 dilution) to each sample. Incubated 5 minutes at 55 degC
  4. Dilute Qiagen protease 1:100 in H2O. Add 1 ul to each sample and incubate 10 minutes at 50 degC, followed by 20 minutes at 70 degC
  5. Make the master mix for Exo- treatment: 1 ul Exo- and 0.4 ul dNTP per reaction
  6. Add 1.4 ul Exo- mix to each sample and incubate 15 minutes at 37 degC, followed by 20 minutes at 75 degC

====# Finish first round PCR master mix (per reaction):

    1. 15 ul 2X KAPA fast SYBR.
    2. 0.6 ul Adapter 1.
    3. 1.4 ul H2O.
  1. Add 0.6 of barcode adapters to each sample
    • Random Primer Mix: 26
    • Oligo (dT)23 VN: 27
    • FISSEQ_RT: 28
    • Top48 Hexamer: 29
    • NTC: 30
    • No Enzyme: 31
    • NxtraPosControl (gDNA): 32
    • NxtraNegControl (H2O): 33
    • NxtraPCRControl (H2O): 34
  2. Add 17 ul of first round PCR master mix to each sample
    • 17ul H2O for NxtraPCRControl
  3. Incubate in thermocycler:
    1. 95 degC for 1 min.
    2. 7 cycles:
      1. 95 degC for 10 sec.
      2. 62 degC for 15 sec.
      3. 72 degC for 45 sec.
  4. Finish second round PCR master mix:
    1. 35 ul KAPA fast SYBR
    2. 2 ul Primer 1
    3. 2 ul Primer 2
    4. 31 ul H2O
  5. Add 70 ul of second round PCR master mix to each sample.
  6. PCR program is as follows:
    1. 95 degC for 1 min.
    2. 10 cycles:
      1. 95 degC for 10 sec.
      2. 60 degC for 15 sec.
      3. 72 degC for 45 sec.
  7. Incubate in thermocycler an additional 3 min @ 72 degC for final extension.

[[]]

  1. Run 5 ul sample on PAGE gel with 3 ul 6X loading dye to identify positives.
    1. 0.5 ul low mass ladder (Invitrogen). Equalize volume with TBE.
    2. 25 min @ 250 V.
    3. Stain 5-10 min in ~100 ml 1X TBE with 6 ul SYBR Safe.

[[]]