Daniel:Notebook/ComboLock/2016-8-29

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Revision as of 16:09, 9 September 2016 by >Djacobse (→‎Protocol)
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Positive Control Dilution Test (Started Friday)

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Protocol

Today I'm testing the samples using the P2 and P12RC amplification primers. Yesterday included the original amplicon materials; this qPCR should be only the DNA still attached to beads.

  1. qPCR X2-Primers
    1. Set up 14 reaction with 2 uL sample, 25 uL KAPA, 22 uL nfH2O, 0.5 uL primer2 and 0.5 uL primer12RC
    2. Load lanes according to table
    3. Sample Name Sample No. qPCR Lane
      Pure C1-C2 Sample 00 A1
      1E+11 Sample 0 B1
      1E+10 Sample 1 C1
      1E+09 Sample 2 D1
      1E+08 Sample 3 E1
      1E+07 Sample 4 F1
      1E+06 Sample 5 G1
      1E+05 Sample 6 H1
      10000 Sample 7 A12
      1000 Sample 8 B12
      100 Sample 9 C12
      10 Sample 10 D12
      1 Sample 11 E12
      NTC NTC F12
    4. Master Mix
      1. 352.5 uL KAPA SyberFast MM
      2. 310.2 uL nfH2O
      3. 7.05 uL primer2
      4. 7.05 uL primer12RC
    5. Add 48 uL Master Mix to each well
    6. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x32
      7. 72C 2 min
      8. 16C hold
  2. TBE Gel
    1. Mix together 72 uL TBE and 18 uL 6X loading dye in a 0.2mL tube
    2. Add 1 or 2 uL sample according to table
    3. Gel Lane Sample Name qPCR Lane uL Sample
      2 25 bp Ladder   2
      3 1E11 Molecule B1 1
      4 1E9 Molecule D1 1
      5 1E7Molecule F1 2
      6 1E5 Molecule H1 2
      7 1000 Molecule B12 2
      8 10 Molecule D12 2
      9 1 Molecule E12 2
    4. Add 10 uL mix to well
    5. Run gel for 25 minutes at 250V
    6. Stain for 3 min with 3 uL SYBR gold
    7. Image in gel doc

Results

X3 Dilution Test

Based on the results from the X2 dilution test, I have a couple of factors I'd like to change for testing X3. From the gel results yesterday, I'd say that I couldn't see signal below 10^9 molecules, which isn't great. However, I'm guessing that this is probably due to loss of bead signal (I lost too many beads). So, I'm going to do 2 things: (1) double the number of beads. This won't actually do anything except make it easier for me to see the beads. The number of beads shouldn't change the results, only the number of targets. (2) Technical replicates. Sometimes n=1 isn't a great idea. So I'll do n=3. And finally, (3rd bonus point!) I'm going to do fewer samples and only test in the higher numbers (0-5 which ranges from 1E11 to 1E6 molecules, respectively).

Buffers

KLN Mix

Reagent Stock uL added
Amp Ligase Buffer 10X 3
dNTPs 100 uM 3
Hemo Klentaq NA 6
Amp Ligase 5 U/uL 3
nfH2O NA 15

Protocol

Tube No (A-C) Dilution Molecules Available
0 1 100000000000
1 10 10000000000
2 100 1.E+09
3 1000 1.E+08
4 10000 1.E+07
5 100000 1.E+06
  1. Latch and Padlock Hybridization
    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 oligo per sample to bead solution; incubate at RT for 5 min
    4. Apply magnet for 30 sec and remove supernatant
    5. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
    6. Add 2 uL correct latch (10 uM stock) and 2 uL 10 uM Padlock0001-68 to each sample to a fresh 0.2 mL tube
    7. Dissolve target in 50 uL wash buffer and heat at 65C for 5 minutes
    8. Chill on ice for 3 minutes
    9. Add sample to prepared strep beads; vortex to suspend
    10. Incubate for 30 min at 37C with intermittent mixing
    11. Apply magnet and remove supernatant
    12. Add 100 uL wash buffer and vortex to suspend beads; apply magnet and remove supernatant
    13. Repeat wash step above
    14. Add 100 uL cold Low Salt Buffer and vortex to suspend; apply magnet and remove supernatant
    15. Resuspend with 20 uL 1x Amp Ligase buffer
  2. Circularization
    1. Prepare 30 uL KLN mix
    2. Add 2 uL KLN mix to 20 uL of reaction
    3. Incubate at 55C overnight; continued tomorrow