Daniel:Notebook/ComboLock/2016-10-27

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Cell Test 3 (Started Friday 10/21)[edit]

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Latch PCR[edit]

Just testing the PCR of Latch0001, which didn't show up at all in the gels from Tuesday. I'm just going to test on the final product, but I'll try to make sure that we get the cells from the bottom of the tubes.

  1. qPCR
    1. Prepare the following master mix
      1. 128.1 uL nfH2O
      2. 6.1 uL primer4
      3. 6.1 uL primer6RC
      4. 152.5 uL Kapa SYBR Fast master mix
    2. Add 48 uL master mix to each well
    3. Add 2 uL sample to correct well by following sample table
    4. Sample Condition Lane Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM p6RC (fwd) Primer 10 uM p4 (rev) Primer H2O Total Volume (uL)
      Sample 1 Exp. Rep 1 A1 2 25 1 1 21 50
      Sample 2 Exp. Rep 2 B1 2 25 1 1 21 50
      Sample 3 No C Probes C1 2 25 1 1 21 50
      Sample 4 No Cells D1 2 25 1 1 21 50
      NTC qPCR primers only E1 0 25 1 1 21 50
      Positve Control Direct Latch0001 (10 uM) F1 2 25 1 1 21 50
    5. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x24
      7. 72C 2 min
      8. 16C hold
  2. TBE Gel
    1. Mix 40 uL TBE, 16 uL 6x loading dye
    2. Aliquot 10 uL per sample/ladder lane onto parafilm
    3. Add 2 uL of sample or ladder to correct drop
    4. Load 10 uL in to well
    5. Run gel for 22 minutes at 250V
    6. Open gel and stain with 3 uL SYBR Gold for 3 minutes
    7. Rinse gel and image in gel doc

Results[edit]

Beads UMI Test[edit]

Test to try and count the number of UMIs detected by qPCR/sequencing. Should be useful for counting the number of successful hybridization events in relation to number of targets present. Ideally, we'll get an idea of hybridization efficiency.

Buffers[edit]

Wash/Binding Buffer

Reagent Stock Final Dilution Amt in 10 mL
NaCl 1.5 M 0.5 M 3 3.33 mL
Tris-HCl 500 mM 20 mM 25 400 uL
EDTA 0.5M 1 mM 500 20 uL
nf H2O NA NA NA 6.25 mL

Low Salt Buffer

Reagent Stock Final Dilution Amt in 10 mL
NaCl 1.5 M 0.15 M 10 1 mL
Tris-HCl 500 mM 20 mM 25 400 uL
EDTA 0.5M 1 mM 500 20 uL
nf H2O NA NA NA 8.58 mL

KLN Mix

Reagent Stock uL added
Amp Ligase Buffer 10X 3.5
dNTPs 100 uM 3.5
Hemo Klentaq NA 7
Amp Ligase 5 U/uL 3.5
nfH2O NA 17.5

Protocol[edit]

Sample C Probes-Positve Control (C1+C2) Template Dilution Dilution Amount
Sample 1 (ABC) X X 0 10 pmol
Sample 2 (ABC) X   NA None
Sample 3 (ABC)   X 0 10 pmol
Sample 4 (ABC) X X 2 100 fmol
Sample 5 (ABC) X X 4 1 fmol
  1. Template-Bead Binding
    1. Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer
    2. Apply magnet for 30 sec and remove supernatant
    3. Add 2 uL 10 uM template oligo per sample to bead solution; incubate at RT for 5 min
    4. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
  2. C Probe Hybridization
    1. Combine 1 uL of each C probe (10 uM stock) and 3 uL bead wash buffer into a 0.2 mL tube
    2. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    3. Add 5 uL probe mixture to beads
    4. Incubate at 40C for 1 hour with agitation
    5. Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
    6. Repeat wash step above
    7. Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant
  3. Latch and Padlock Hybridization
    1. Add 2 uL correct latch (10 uM stock) and 2 uL 10 uM Padlock0201 to each sample to a fresh 0.2 mL tube
      1. Tech Rep A-Latch0001
      2. Tech Rep B-Latch0002
      3. Tech Rep C-LatchX04
    2. Heat at 65C for 5 minutes
    3. Chill on ice for 3 minutes
    4. Add sample to prepared strep beads; vortex to suspend
    5. Incubate for 30 min at 37C with intermittent mixing
    6. Apply magnet and remove supernatant
    7. Add 100 uL wash buffer and vortex to suspend beads; apply magnet and remove supernatant
    8. Repeat wash step above
    9. Add 100 uL cold Low Salt Buffer and vortex to suspend; apply magnet and remove supernatant
    10. Resuspend with 20 uL 1x Amp Ligase buffer
  4. Circularization
    1. Prepare 10 uL KLN mix
    2. Add 2 uL KLN mix to 20 uL of reaction
    3. Incubate at 55C overnight; continued tomorrow