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User:DrJones1935/30 July 2012
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(Created page with "====I. Prepare for Gel Extraction of a PCR Product from 27 Jul 2012==== *'''Make Gel:''' :*Measure out 0.75 g of agarose and add to a 250 mL E-flask :*Add 75 mL of 0.5x TBE buf...") |
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====IV. Image Post-Stained Portion of Gel==== | ====IV. Image Post-Stained Portion of Gel==== | ||
Latest revision as of 01:50, 4 October 2012
Contents |
I. Prepare for Gel Extraction of a PCR Product from 27 Jul 2012
- Make Gel:
- Measure out 0.75 g of agarose and add to a 250 mL E-flask
- Add 75 mL of 0.5x TBE buffer and swirl to mix
- Cover flask with a Kimwipe and microwave for ~1 minute until clear
- Allow to cool to ~60 oC
- Immediately pour gel into tray with combs
- Allow to solidify, then remove the comb and tape and place the tray in the gel box with the wells closer to the black electrode
- Make sure it is submerged in TBE buffer
- Note: Some gel leaked out the ends
- Load Gel:
- Mix sample in PCR tubes with 10 uL buffer. Load 40 uL into one lane and the rest into another.
- Load on gel according to the following chart:
| Lane 1 | 2 | 3 |
|---|---|---|
| Invitrogen 1kb+ ladder | +1 (~10 uL) | +1 (~40 ul) |
- Run Gel:
- Close gel box and turn on power pack
- Run gel at 30 V for a while to stack gel (while at lunch)
- Run gel at 75 V until the markers have reached ~3/4 down the gel
- Cut Gel:
- Remove gel from box and cut along lanes to separate the 10 uL lane from the 40 uL lane.
- Return 40 uL lane to the gel box
- Post-stain with EtBr
- Place 10 uL lane in 200 mL of 0.5 ug/mL EtBr solution for 15 minutes
- Remove EtBr solution and wash with 200 mL of dH2O for 10 minutes
- Image Gel
- View 10 uL lane under UV to identify band of interest
- Use a clean razor blade to nick gel where the band is
- Excise Gel Fragments
- Match 40 uL lane to the 10 uL lane
- Using the 10 uL lane as a guide, cut out the band from the 40 uL lane
II. QIAquick Gel Extraction Protocol
- Weigh each gel slice in a colorless tube:
| Cassette (+1) |
|---|
| 389 mg |
- Add 3 volumes Buffer QG to 1 volume of gel
| Cassette (+1) |
|---|
| 1167 uL |
- Incubate in 50 oC water bath for 10-12 minutes until all gel has dissolved. Vortex to help mix.
- Note: The solutions were all yellow, OK to proceed
- Add 1 volume 100% isopropanol to samples and mix by inversion
| Cassette (+1) |
|---|
| 389 uL |
- Apply sample to a QIAquick column 800 uL at a time. Centrifuge for 1 minute at 13000 rpm, discard flow-through, and repeat until all of the sample has passed through the column
- Add 500 uL of Buffer QG to the QIAquick columns and centrifuge for 1 minute, discard flow-through
- Add 750 uL of Buffer PE to the QIAquick columns and let the column stand for 5 minutes on the bench
- Centrifuge for 1 min and discard flow through
- Centrifuge again for 1 minute
- Transfer the columns to clean microcentrifuge tubes
- Elute DNA by adding 30 uL of Buffer EB to the center of the column and let stand for 4 minutes
- Centrifuge for 1 minute
III. Measure the Concentration of Extracted DNA
- Bring sample and Buffer EB bottle upstairs to Take3 plate reader
- Add 2 uL of each sample according to the following chart:
| Blank (EB) | Blank |
| Blank | Blank (diverging, ignored) |
| Cassette | (empty) |
- Use the program to blank the machine and measure sample concentrations
RESULTS
Concentration:
| Sample | Concentration (ng/uL) | Approx. volume (uL) | Approx. total (ug) |
|---|---|---|---|
| Cassette | 0 | ~28 | 0 |
IV. Image Post-Stained Portion of Gel
RESULTS
Source: Media:Srk_2012-07-30_15hr_02min.tif
| Feature | Expected Size (bp) |
|---|---|
| Cassette | 5152 |
