User:DrJones1935/23 July 2012
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Revision as of 23:27, 3 October 2012
Contents |
I. Run Diagnostic PCR on Sample 3
- Mix Reagents:
- Mix according to the following table
A | B | C | D | E | F | |
---|---|---|---|---|---|---|
Reagent | (uL) | (uL) | (uL) | (uL) | (uL) | (uL) |
water | 33.5 | 34.5 | 36 | 36 | 33.5 | 34 |
5x Phusion HiFi buffer | 10 | 10 | 10 | 10 | 10 | 10 |
10 mM dNTP mix | 1 | 0 | 1 | 1 | 1 | 1 |
10 uM primer (F) | 2.5 (p3f_2) | 2.5 (p3f_2 | 0 | 2.5 (p3f_2) | 2.5 (p3f_2) | 2.5 (p3f_2) |
10 uM primer (R) | 2.5 (p3r) | 2.5 (p3r) | 2.5 (p3r) | 0 | 2.5 (p3r) | 2.5 (p3r) |
Template | ECNR2 colony* | ECNR2 colony* | ECNR2 colony* | ECNR2 colony* | 0 | ECFI5 colony* |
Phusion HiFi polymerase | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0 |
*For these templates, I touched a colony with a sterile pipette tip and spread it on the bottom of the PCR tube before adding reagents
- Run PCR
Step | Temp (oC) | Time |
---|---|---|
1 | 94 | 4 m |
2 | 94 | 30 s |
3 | 53 | 30 s |
4 | 72 | 1.5 m |
5 | GOTO 2 | 7x |
6 | 94 | 30 s |
7 | 66 | 30 s |
8 | 72 | 1.5 m |
9 | GOTO 6 | 30x |
10 | 72 | 5 m |
11 | 4 | ∞* |
- Store tubes on ice after PCR is finished
II. Prepare for Gel Extraction of PCR Products from 21 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
- Load Gel:
- Mix samples in PCR tubes with 10 uL buffer. Load 40 uL into one lane and the rest into another.
- Load ~6 uL on gel according to the following chart:
Lane 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
---|---|---|---|---|---|---|---|
NEB 2-log ladder | 2.2 (~10 uL) | 2.2 (~40 ul) | NEB 2-log ladder | 3.2 (~10 uL) | 3.2 (~40 uL) | 4.1 (~10 uL) | 4.1 (~40 uL) |
- Store DNA at 4 oC
- 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 down the gel
- Cut Gel:
- Remove gel from box and cut along lanes to separate the 10 uL lanes from the 40 uL lanes.
- Return 40 uL lanes to the gel box
- Post-stain with EtBr
- Place 10 uL lanes 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 lanes under UV to identify bands of interest
- Use a clean razor blade to nick gel where the bands are
- Excise Gel Fragments
- Match 40 uL lanes to the 10 uL lanes
- Using the 10 uL lanes as a guide, cut out the bands from the 40 uL lanes
III. QIAquick Gel Extraction Protocol
- Weigh each gel slice in a colorless tube:
LacZ (2.2) | CAT* (3.2) | tetr <4.1) |
---|---|---|
233 mg | 263 mg | 409 mg (treated as 400 mg) |
- Add 3 volumes Buffer QG to 1 volume of gel
LacZ (2.2) | CAT* (3.2) | tetr <4.1) |
---|---|---|
699 uL | 789 uL | 1200 uL |
- Incubate in 50 oC water bath for 10-15 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
LacZ (2.2) | CAT* (3.2) | tetr <4.1) |
---|---|---|
233 uL | 263 uL | 409 uL |
- Apply each 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 5 minutes
- Centrifuge for 1 minute
IV. Measure the Concentration of Extracted DNA
- Bring samples 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) |
LacZ | CAT* |
tetR | (empty) |
- Use the program to blank the machine and measure sample concentrations
RESULTS
File: Media:srk_gel_extraction_23jul12.xlsx
Concentration:
Plasmid | Concentration (ng/uL) | Approx. volume (uL) | Approx. total (ug) |
---|---|---|---|
LacZ | 11.468 | ~28 | 0.321 |
CAT* | 35.256 | ~28 | 0.987 |
tetR | 37.787 | ~28 | 1.058 |
V. AGE of Diagnostic PCR samples
- Make Gel:
- Measure out 0.5 g of agarose and add to a 250 mL E-flask
- Add 50 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 and pour into flask 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 fresh TBE buffer
- Load Gel:
- Mix samples as drops on a piece of parafilm
- Mix:
Ladder (NEB 2-log) | Blank | Sample |
---|---|---|
*5 uL DNA ladder *1 uL 6x Loading Dye | *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 ~8 uL (except ladder which is ~6 uL) on gel according to the following chart:
Lane 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 |
---|---|---|---|---|---|---|---|---|---|---|
NEB 2-log ladder | empty | Blank | A | empty | B | empty | C | D | E | F |
- Store DNA at 4 oC
- Unusual loading pattern was due to poor loading
- Run Gel:
- Close gel box and turn on power pack
- Run gel at 30 V for 20 min to stack gel
- Run gel at 75 V until the markers have reached ~halfway down gel
- Image Gel:
- RESULTS:
Source: Media:Srk_2012-07-23_21hr_46min.tif