User:DrJones1935/23 July 2012

From 2012.igem.org

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

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:
Srk 2012-07-23 21hr 46min.jpg

Source: Media:Srk_2012-07-23_21hr_46min.tif