Daniel:Notebook/ComboLock/2017-3-3: Difference between revisions
Jump to navigation
Jump to search
>Djacobse |
>Djacobse |
||
Line 54: | Line 54: | ||
===Results=== | ===Results=== | ||
<gallery perrow=3 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 |
Revision as of 22:51, 6 March 2017
Padlock0601 Test (Started Yesterday)
Protocol-Part 5
- TBE Gel
- Mix 240 uL TBE, 60 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 23 minutes at 230V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
- qPCR
- Make 9.2X qPCR master mix according to following recipe
- 193.2 uL nfH2O
- 230 uL SYBR Fast
- 9.2 uL 10 uM AmpF
- 9.2 uL 10 uM AmpR-Ind20
- Add 48 uL master mix to each well
- Add 2 uL sample according to plate layout File:PlateLayout-20170303-Padlock0601-Bglii.png
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x30
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 96 uL TBE, 24 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 23 minutes at 230V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Results
- 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
- 20170302-qPCR-Padlock0601Test.png
qPCR curves for run on 3-2-2017
- 2017-03-03-Padlock0601-PreRCA-Unpurifed.png
Gel image-PreRCA Unpurified
- 2017-03-03-Padlock0601-PreRCA-Purifed.png
Gel image-PreRCA Purifed
- 2017-03-03-Padlock0601-PostRCA-Unpurifed.png
Gel image-PostRCA Unpurified
- 2017-03-03-Padlock0601-PostRCA-Purifed.png
Gel image-PostRCA Purified
- 20170303-qPCR-Padlock0601-Bglii.png
qPCR curves for post-Bglii digestion
- 2017-03-03-Padlock0601-Bglii.png
Post BglII digestion