Daniel:Notebook/ComboLock/2017-1-25: Difference between revisions

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===Library Prep Protocol===


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Revision as of 21:37, 26 January 2017

Circularization Test (Started Monday)

Back to Calendar

Protocol-New Amplification Primers

These are new primers I designed. They are slightly adjusted from the locations of AmpF/AmpR. Hopefully there is less noise.

  1. qPCR
    1. Make qPCR master mix according to following recipe
      1. 361.2 uL nfH2O
      2. 17.2 uL AmpF-CLv3
      3. 17.2 uL AmpR-CLv3
      4. 430 uL Kapa SYBR Fast
    2. Add 48 uL master mix to each well
    3. Add 2 uL sample according to table
    4. Sample Product Amt Pre/Post RCA Lane Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Total Volume (uL)
      Sample 0A 1pmol Pre A1 2 25 1 1 21 50
      Sample 0B 1pmol Pre A2 2 25 1 1 21 50
      Sample 2A 10 fmol Pre A3 2 25 1 1 21 50
      Sample 2B 10 fmol Pre A4 2 25 1 1 21 50
      Sample 4A 100 amol Pre A5 2 25 1 1 21 50
      Sample 4B 100 amol Pre A6 2 25 1 1 21 50
      Sample 6A 1 amol Pre A7 2 25 1 1 21 50
      Sample 6B 1 amol Pre A8 2 25 1 1 21 50
      Sample 0AX 1pmol Post H1 2 25 1 1 21 50
      Sample 0BX 1pmol Post H2 2 25 1 1 21 50
      Sample 2AX 10 fmol Post H3 2 25 1 1 21 50
      Sample 2BX 10 fmol Post H4 2 25 1 1 21 50
      Sample 4AX 100 amol Post H5 2 25 1 1 21 50
      Sample 4BX 100 amol Post H6 2 25 1 1 21 50
      Sample 6AX 1 amol Post H7 2 25 1 1 21 50
      Sample 6BX 1 amol Post H8 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 80 uL TBE, 20 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

RCA with Epicentre Phi29

Still have some left, so I'm going to test its effectiveness by trying to amplify the 1 amol sample. I'll use the original Epicentre concentration and a 10X dilution, since the Epicentre Phi29 is 10X more concentrated than NEB.

Sample Matrix

Sample Condition
Sample 6AC Epicentre-1:10
Sample 6BC Epicentre-1:10
Sample 6AD Epicentre
Sample 6BD Epicentre
  1. Rolling Circle Amplification
    1. Prepare 7X Master mix according to table below
    2. Add 15 uL master mix to each tube
    3. Add sample to new reaction tubes according to following table
    4. Reagent uL Added Master Mix (4.5X)
      Template 5 NA
      RCA Primer (LLRC 10 uM) 2.5 11.25
      dNTP (1 mM) 0.8 3.6
      10X Buffer 2 9
      Phi29 1 0
      nfH2O 8.7 39.15
      Total 20 63
    5. Incubate at 37C for 3 hours
    6. Incubate at 65C for 10 minutes;Hold at 10C until next step

    Continued tomorrow

    Library Prep

    Based on yesterday's results, I'm going to sequence the most successful lanes (PreRCA-1pmol; PostRCA-1 pmol, 10 fmol, and 100 amol). With the UMI now on the padlock, this should give a relative percentage of padlock capture. Each sample has a unique AmpR index.

    Sample Matrix

    Sample Sample Amt Pre/Post RCA AmpR Index
    Sample 0 (AB) 1 pmol Pre 21
    Sample 0X (AB) 1 pmol Post 25
    Sample 2X (AB) 10 fmol Post 26
    Sample 4X (AB) 100 amol Post 27
    1. Size Select Gel
      1. Mix 20 uL sample from each tech rep lane (A and B) and 8 uL 6x loading dye into separate tubes for each sample (Samples 1A and 1B)
      2. Mix 4 uL ladder, 4 uL 6x dye, and 16 uL TBE in ladder tube
      3. Aliquot 48/24 uL per sample/ladder lane into each well
      4. Run gel for 23 minutes at 230V
      5. Open gel and stain with 3 uL SYBR Gold for 3 minutes
      6. Rinse gel and image in gel doc
      7. Cut out appropriate bands with scalpel; put in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube
      8. Image in gel doc
      9. Centrifuge at 12000 rpm for 1.5 minutes
      10. Add 500 uL TE to each sample
      11. Incubate 4 hours at 37C with vigorous shaking;
      12. Centrifuge tubes at 12000 rpm for 1.5 minutes to bring gel to bottom
      13. Extract supernatant and add to nanosep column
      14. Centrifuge at 12000 rpm for 1.5 minutes; collect supernatant
    2. Ethanol Precipitation
      1. Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 1.5 uL glycoblue to each sample
      2. Incubate at -80C overnight; continued tomorrow