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User:DrJones1935/8 August 2012
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(Created page with "====I. Prepare for Gel Extraction of LacZ Product from HF2==== *'''Make Gel:''' :*Measure out 0.40175 g of agarose and add to a 250 mL E-flask :*Add 50 mL of 0.5x TBE buffer and...")
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(Created page with "====I. Prepare for Gel Extraction of LacZ Product from HF2==== *'''Make Gel:''' :*Measure out 0.40175 g of agarose and add to a 250 mL E-flask :*Add 50 mL of 0.5x TBE buffer and...")
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Revision as of 23:35, 3 October 2012
Contents |
I. Prepare for Gel Extraction of LacZ Product from HF2
- Make Gel:
- Measure out 0.40175 g of agarose and add to a 250 mL E-flask
- Add 50 mL of 0.5x TBE buffer and swirl to mix (0.8% gel)
- Cover flask with a Kimwipe and microwave for ~1 minute until clear
- Allow to cool to ~60 oC
- Immediately pour gel into tray with modified combs (3 taped together for large well)
- 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 fresh TBE buffer
- Load Gel:
- Mix:
| Ladder (NEB 2-log) | 10 uL well | 42 uL well |
|---|---|---|
| *4 uL pre-mixed | *6.3 uL dH2O *1.7 uL 6x Loading Dye *2.0 uL DNA | *6 uL 6x Loading Dye *36 uL DNA |
- Load on gel according to the following chart:
| Lane 1 | 2 | 3-5 |
|---|---|---|
| NEB 2-log ladder | LacZ (~10 uL) | LacZ (~45 ul) |
- Run Gel:
- Close gel box and turn on power pack
- Run gel at 30 V for 20 min
- Run gel at 75 V until the markers have reached 3/4 of gel
- Cut Gel:
- Remove gel from box and cut along lanes to separate the 10 uL lane from the 45 uL lane.
- Return 45 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
- 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 45 uL lane to the 10 uL lane
- Using the 10 uL lane as a guide, cut out the band from the 45 uL lane
II. QIAquick Gel Extraction Protocol
- Weigh each gel slice in a colorless 15 mL conical tube:
| LacZ (HF2) |
|---|
| 275 mg |
- Add 3 volumes Buffer QG to 1 volume of gel
| LacZ (HF2) |
|---|
| 825 uL |
- Incubate in 50 oC water bath for 12 minutes until all gel has dissolved. Vortex to help mix.
- Note: The solution was yellow, OK to proceed
- Add 1 volume 100% isopropanol to samples and mix by inversion
| LacZ (HF2) |
|---|
| 275 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 column and centrifuge for 1 minute, discard flow-through
- Add 750 uL of Buffer PE to the QIAquick column, 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 |
| LacZ | (empty) |
- Use the program to blank the machine and measure sample concentrations
RESULTS
File: Media:srk_gel_extraction_8aug12.xlsx
Concentration:
| Sample | Concentration (ng/uL) | Approx. volume (uL) | Approx. total (ug) |
|---|---|---|---|
| LacZ | 77.185 | ~28 | 2.16 |
IV. Image Post-Stained Gel
RESULTS
Source: Media:Srk_2012-08-08_13hr_42min.tif
| Feature | Expected Size (bp) |
|---|---|
| LacZ | 3151 |
V. Cross-over PCR
- Template Mix Specifications:
Mix I:
| Template | Size (bp) | Amount Wanted (pmol) | Amount Wanted (ng) | Concentration (ng/uL) | Volume (1x) | Volume (5x) |
|---|---|---|---|---|---|---|
| LacZ | 3151 | 0.01 | 20.45 | 77.185 | 0.26 | 1.32 |
| CAT* | 729 | 0.01 | 4.73 | 35.256 | 0.13 | 0.67 |
| tetR | 1265 | 0.01 | 8.21 | 37.787 | 0.22 | 1.09 |
| total | 3.08 uL |
Mix II:
| Template | Size (bp) | Amount Wanted (pmol) | Amount Wanted (ng) | Concentration (ng/uL) | Volume (1x) | Volume (5x) |
|---|---|---|---|---|---|---|
| LacZ | 3151 | 0.05 | 102.25 | 77.185 | 1.32 | 6.62 |
| CAT* | 729 | 0.05 | 23.65 | 35.256 | 0.67 | 3.35 |
| tetR | 1265 | 0.05 | 41.05 | 37.787 | 1.09 | 5.45 |
| total | 15.42 uL |
- *Calculations made with [http://molbiol.edu.ru/eng/scripts/h01_07.html this website]
- Mix Reagents:
- Mix according to the following table
Mix I:
| 1 Reaction | Master Mix (x6) | |
|---|---|---|
| Reagent | (uL) | (uL) |
| water | 32.384 | 194.3 |
| 5x KAPA HiFi buffer | 10 | 60 |
| 10 mM dNTP mix | 1.5 | 9 |
| 10 uM primer (F) | 2.5 (pBf) | 15 (pBf) |
| 10 uM primer (R) | 2.5 (pBr) | 15 (pBr) |
| Template Mix | 0.616 | - |
| KAPA HiFi polymerase | 0.5 | 3 |
Mix II:
| 1 Reaction | Master Mix (x6) | |
|---|---|---|
| Reagent | (uL) | (uL) |
| water | 29.916 | 179.496 |
| 5x KAPA HiFi buffer | 10 | 60 |
| 10 mM dNTP mix | 1.5 | 9 |
| 10 uM primer (F) | 2.5 (pBf) | 15 (pBf) |
| 10 uM primer (R) | 2.5 (pBr) | 15 (pBr) |
| Template Mix | 3.084 | - |
| KAPA HiFi polymerase | 0.5 | 3 |
- Before adding template, move 49.384 uL of Master Mix I to a clean PCR tube and add 0.616 uL water for negative control
- Before adding template, move 46.916 uL of Master Mix II to a clean PCR tube and add 3.084 uL water for negative control
- Add templates to Master Mixes according to Template Specifications above, mix by pipetting
- Aliquot 50 uL of each mix to 5 PCR tubes each
- Note: Last tube for each had less than others
- Run PCR
| Step | Temp (oC) | Time |
|---|---|---|
| 1 | 94 | 4 m |
| 2 | 94 | 30 s |
| 3 | 51 | 30 s |
| 4 | 72 | 5 m |
| 5 | GOTO 2 | 7x |
| 6 | 94 | 30 s |
| 7 | 63 | 30 s |
| 8 | 72 | 5 m |
| 9 | GOTO 6 | 30x |
| 10 | 72 | 5 m |
| 11 | 4 | ∞ |
VI. AGE of PCR Samples
- Make Gel:
- Measure out 0.40228 g of agarose and add to a 250 mL E-flask
- Add 50 mL of 0.5x TBE buffer and swirl to mix (0.8% gel)
- Cover flask with a Kimwipe and microwave for ~1 minute until clear
- Allow to cool to ~60 oC and pour into beaker for ethidium bromide (EtBr)
- Add 1 uL of EtBr stock to agarose and swirl to mix
- 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
- Load Gel:
- Mix samples as drops on a piece of parafilm
- Mix:
| Ladder (Invtrogen 1 kb plus) | Blank | Sample |
|---|---|---|
| *4 uL pre-mixed | *8.3 uL dH2O *1.7 uL 6x Loading Dye | *6.3 uL dH2O *1.7 uL 6x Loading Dye *2.0 uL DNA |
- Load ~10 uL on gel according to the following chart (except ladder which is 6 uL):
| Lane 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| I- | I1 | I2 | I3 | I4 | I5 | 2-log Ladder | II- | II1 | unusable | II2 | II3 | II4 | II5 |
- Store DNA at 4 oC
- Unusual well formation in some cases
- Run Gel:
- Close gel box and turn on power pack
- Run gel at 30 V for 25 min to stack gel
- Run gel at 75 V until the markers have reached ~3/4 of gel
- Image Gel:
- RESULTS:
Source: Media:Srk_2012-08-09_00hr_14min.tif
| Feature | Expected Size (bp) |
|---|---|
| Cassette | 5152 |
- Arrow indicates desired product
