Daniel:Notebook/ComboLock/2017-3-3: Difference between revisions

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=Padlock0601 Test (Started [[Daniel:Notebook/ComboLock/2017-3-1|Yesterday]])=
=Padlock0601 Test (Started [[Daniel:Notebook/ComboLock/2017-3-1|Wednesday]])=


[[Daniel:Notebook/ComboLock|Back to Calendar]]
[[Daniel:Notebook/ComboLock|Back to Calendar]]
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===Results===
===Results===


<gallery perrow=2 heights=300px widths=300px>
<gallery perrow=3 heights=300px widths=300px caption="qPCR curves and gel images for samples. See individual gel captions for more info. Note that for all gel images Post-RCA the left sample was RC amplified with normal primer and the right sample with phosphothiorate primer">
File:20170302-qPCR-Padlock0601Test.png|qPCR curves for run on 3-2-2017  
File:20170302-qPCR-Padlock0601Test.png|qPCR curves for run on 3-2-2017  
File:2017-03-03-Padlock0601-PreRCA-Unpurifed.png|Gel image-PreRCA Unpurified
File:2017-03-03-Padlock0601-PreRCA-Unpurifed.png|Gel image-PreRCA Unpurified
Line 60: Line 60:
File:2017-03-03-Padlock0601-PostRCA-Unpurifed.png|Gel image-PostRCA Unpurified
File:2017-03-03-Padlock0601-PostRCA-Unpurifed.png|Gel image-PostRCA Unpurified
File:2017-03-03-Padlock0601-PostRCA-Purifed.png|Gel image-PostRCA Purified
File:2017-03-03-Padlock0601-PostRCA-Purifed.png|Gel image-PostRCA Purified
File:20170303-qPCR-Padlock0601-Bglii.png|qPCR curves for post-Bglii digestion
File:2017-03-03-Padlock0601-Bglii.png|Post BglII digestion
File:2017-03-03-Padlock0601-Bglii.png|Post BglII digestion
</gallery>
</gallery>

Latest revision as of 23:03, 6 March 2017

Padlock0601 Test (Started Wednesday)[edit]

Back to Calendar

Protocol-Part 5[edit]

  1. TBE Gel
    1. Mix 240 uL TBE, 60 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 23 minutes at 230V
    6. Open gel and stain with 3 uL SYBR Gold for 3 minutes
    7. Rinse gel and image in gel doc
  2. qPCR
    1. Make 9.2X qPCR master mix according to following recipe
      1. 193.2 uL nfH2O
      2. 230 uL SYBR Fast
      3. 9.2 uL 10 uM AmpF
      4. 9.2 uL 10 uM AmpR-Ind20
    2. Add 48 uL master mix to each well
    3. Add 2 uL sample according to plate layout
    4. File:PlateLayout-20170303-Padlock0601-Bglii.png
    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 x30
      7. 72C 2 min
      8. 16C hold
  3. TBE Gel
    1. Mix 96 uL TBE, 24 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 23 minutes at 230V
    6. Open gel and stain with 3 uL SYBR Gold for 3 minutes
    7. Rinse gel and image in gel doc

Results[edit]