Daniel:Notebook/ComboLock/2017-1-19: Difference between revisions

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===Discussion (RCA Test)===
From the results on the table, I'd say there was amplification (on the order of 35X for sample 1) with AmpF '''and''' AmpR. Based on the potential dirtiness of the AmpF and AmpR primers, I'm going to repeat the experiment tomorrow using my adapter primers.
Continued [[Daniel:Notebook/ComboLock/2017-1-20|tomorrow]]


[[Category:ComboLock]] [[Category:20170113]]
[[Category:ComboLock]] [[Category:20170113]]

Latest revision as of 18:37, 20 January 2017

Version 3 Oligos (Started Thursday, Jan 13)[edit]

Back to Calendar

RCA Repeat (Started Yesterday)[edit]

  1. qPCR
    1. Make qPCR master mix according to following table
    2. Master Mix Primer Pair Forward Primer (10 uM) uL Reverse Primer (10 uM) uL uL nfH2O uL Kapa SYBR Fast Master Mix 2X
      MM1 AmpF/AmpR 9.2 NA 193.2 230
      MM2 Primer12RC/AmpRInd25 9.2 9.2 193.2 230
    3. Add 47 (MM1) or 48 (MM2) uL master mix to each well
    4. Add 2 uL sample and 1 uL appropriate Reverse Primer (10 uM, MM1 only) according to table
    5. Sample Condition Lane Primer Pair AmpR Index Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Total Volume (uL) Master Mix
      Sample 1A Sample A1 AmpF/AmpR 21 2 25 1 1 21 50 MM1
      Sample 1B Sample A2 AmpF/AmpR 21 2 25 1 1 21 50 MM1
      Sample 2A No Template A3 AmpF/AmpR 22 2 25 1 1 21 50 MM1
      Sample 2B No Template A4 AmpF/AmpR 22 2 25 1 1 21 50 MM1
      Sample 3A No C Probe A5 AmpF/AmpR 23 2 25 1 1 21 50 MM1
      Sample 3B No C Probe A6 AmpF/AmpR 23 2 25 1 1 21 50 MM1
      Sample 4A No PCCB Phosphate A7 AmpF/AmpR 24 2 25 1 1 21 50 MM1
      Sample 4B No PCCB Phosphate A8 AmpF/AmpR 24 2 25 1 1 21 50 MM1
      Sample 1A Sample-PostRCA H1 Primer12RC/AmpR 25 2 25 1 1 21 50 MM2
      Sample 1B Sample-PostRCA H2 Primer12RC/AmpR 25 2 25 1 1 21 50 MM2
      Sample 4A No PCCB Phosphate H3 Primer12RC/AmpR 25 2 25 1 1 21 50 MM2
      Sample 4B No PCCB Phosphate H4 Primer12RC/AmpR 25 2 25 1 1 21 50 MM2
      Sample 1A Sample-PreRCA H5 Primer12RC/AmpR 25 2 25 1 1 21 50 MM2
      Sample 1B Sample-PreRCA H6 Primer12RC/AmpR 25 2 25 1 1 21 50 MM2
      Sample 4A No PCCB PO4-PreRCA H7 Primer12RC/AmpR 25 2 25 1 1 21 50 MM2
      Sample 4B No PCCB PO4-PreRCA H8 Primer12RC/AmpR 25 2 25 1 1 21 50 MM2
      NTC qPCR Primers Only D1 AmpF/AmpR 26 2 25 1 1 23 50 MM1
      NTC qPCR Primers Only D2 Primer12RC/AmpR 25 2 25 1 1 23 50 MM2
    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 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[edit]

Discussion[edit]

Ummmm. So the AmpR/Primer12RC should only amplify circular product (yesterday's p12/p24 combo is good for circular product and bridge). So once again it showed up in both pre and post RCA reactions, but the normal AmpR/AmpF pairing (necessary for sequencing) showed nothing. Also, assuming I didn't mix up the samples, it kinda seems like there is more product Pre-RCA than post. Going off the qPCR curves (generally a bad idea but probably comparable between these two) it looks like the "PreRCA" sample fraction is ~4X more than the "PostRCA" fraction. This actually makes a ton of sense if there was no amplification from RCA. Both PCRs had 2 uL sample added to them, but the "PostRCA" fraction got 5 uL (out of 20) of "PreRCA" fraction as input. So basically I put the "PreRCA" fraction into water, or might as well have.

RCA Test[edit]

Since it seems like its RCA that is the problem I will do RCA again, this time testing both AmpF and AmpR as (dirty) primers. To save on sample space, I'll use 1 and 4 again (4 is a suitable negative control). I should know whether or not the product amplifies in even half an hour, so we're gonna go with that (measure using Qubit)

  1. Rolling Circle Amplification
    1. Prepare 8.5X 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 (8.5X)
      Template 5 NA
      RCA Primer (AmpF or AmpRInd21; 10 uM) 2.5 21.25
      dNTP (1 mM) 0.8 6.8
      10X Phi29 Buffer 2 17
      Phi29 1 8.5
      nfH2O 8.7 73.95
      Total 20 127.5
    5. Incubate at 37C for 30 minutes
    6. Incubate at 65C for 10 minutes;Hold at 10C until next step
  2. Qubit Measure
    1. Mix dye to Qubit buffer; 10 uL dye in 1990 uL buffer (1:200 ratio per sample)
    2. Add 190 uL dye/buffer and 10 uL standard to standard tubes
    3. Add 199 uL dye/buffer and 1 uL sample to sample tubes
    4. Measure in Qubit

Results[edit]

Sample Name 1A 1B 1AY 1BY 1AF 1BF 4AF 4BF 1AR 1BR 4AR 4BR
Sample Condition Sample-PreRCA Sample-PreRCA Sample Sample Sample Sample No Phosphate (PCCB) No Phosphate (PCCB) Sample Sample No Phosphate (PCCB) No Phosphate (PCCB)
RCA Primer None None LLRC (Mine) LLRC (Mine) AmpF AmpF AmpF AmpF AmpR AmpR AmpR AmpR
Conc (ng/uL) 0.81 1.27 0.72 0.62 35.1 46.9 35 40.7 25.3 26.8 31.5 30.2

Discussion (RCA Test)[edit]

From the results on the table, I'd say there was amplification (on the order of 35X for sample 1) with AmpF and AmpR. Based on the potential dirtiness of the AmpF and AmpR primers, I'm going to repeat the experiment tomorrow using my adapter primers.

Continued tomorrow