User:DrJones1935/3 August 2012

From 2012.igem.org

I. AGE of PCR Samples

  • Make Gel:
  • Measure out 0.40136 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% agarose 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) Sample
*5 uL DNA ladder
*1 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
NEB 2-log ladder - PP 2 PP 5 PP 10 GE 2 GE 5 GE 10
  • Store DNA at 4 oC
  • 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 bottom of the gel
  • Image Gel:
  • RESULTS:
No observable product in any lanes

Source: Media:Srk_2012-08-03_14hr_12min.tif

Feature Expected Size (bp)
Cassette 5152


II. Cross-over PCR

  • Template Mix Specifications:
Template Size (bp) Amount Wanted (pmol) Amount Wanted (ng) Concentration (ng/uL) Volume (1x) Volume (5x)
LacZ 3151 0.00745 15.24 11.468 1.33 6.7
CAT* 729 0.00745 3.52 35.256 0.1 0.5
tetR 1265 0.00745 6.12 37.787 0.16 0.8
total 8 uL
  • Mix Reagents:
  • Mix according to the following table
1 Reaction Master Mix (x6)
Reagent (uL) (uL)
water 31.4 188.4
5x KAPALongRange 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 1.6 -
KAPA LongRange polymerase 0.5 3
  • Before adding template, move 48.4 uL of Master Mix to a clean PCR tube and add 1.6 uL water for negative control
  • Add templates to Master Mix according to Template Specifications above, mix by pipetting
  • Aliquot 50 uL of mix to 5 PCR tubes
  • Run PCR O/N
Step Temp (oC) Time
1 94 4 m
2 94 30 s
3 54 30 s
4 68 5 m
5 GOTO 2 7x
6 94 30 s
7 66 30 s
8 68 5 m
9 GOTO 6 30x
10 72 5 m
11 4



III. Streak out ECNR2 Plate

  • Streak ECNR2 onto an LB plate from glycerol stock
  • Place in 34 oC static incubator overnight