Team:Kyoto/Measurement

From 2011.igem.org

(Difference between revisions)
(→Measurement)
Line 42: Line 42:
#Centrifuge these tubes again and discarde the supernatant and added 1.2ml media(-casamino acids) at 37 ℃.
#Centrifuge these tubes again and discarde the supernatant and added 1.2ml media(-casamino acids) at 37 ℃.
#Bring up E.coli at 0,5,10,15,20,25,30,60min and extracted RNA and synthesized cDNA.
#Bring up E.coli at 0,5,10,15,20,25,30,60min and extracted RNA and synthesized cDNA.
 +
 +
 +
===='''preliminary experiments'''====
 +
 +
'''1-1:plotting the standard line'''
 +
 +
 +
{|
 +
|Sample
 +
  |  glucose solution(various concentration from 0.20 mM to 1.60 mM)
 +
|-
 +
|Blank
 +
  |  mixed liquid (240μℓDNS reagent plus 1760㎕distilled water　)
 +
|}
 +
 +
 +
#added 240㎕ DNS reagent to 80㎕sample
 +
#heated it for 5 min in boiling water and then cooled it in water.
 +
#added it distilled water by 2ｍｌ
 +
#measured absorbance in 550nm
 +
 +
The standard curve is shown in fig 1.
 +
 +
 +
 +
 +
'''1-2:evaluate the affection of medium'''
 +
 +
we examined the interruption based on the components of media(SOC, plas-grow and M9)
 +
 +
 +
{|
 +
|Sample
 +
  |  each medium
 +
|-
 +
|Blank
 +
  |  mixed liquid (240μℓDNS reagent plus 1760㎕distilled water　)
 +
|}
 +
 +
 +
#added 240㎕ DNS reagent to 80㎕sample
 +
#heated it for 5 min in boiling water and then cooled it in water.
 +
#added it distilled water by 2ｍｌ
 +
#measured absorbance in 550nm
 +
 +
The result is shown in fig 2.
 +
 +
'''1-3:measurement the effect of remainded E.coli'''
 +
 +
 +
{|
 +
|Sample
 +
  |  each medium
 +
|-
 +
|Blank
 +
  |  mixed liquid (240μℓDNS reagent plus 1760㎕distilled water　)
 +
|}
 +
 +
 +
This assay was performed three times for each medium   
 +
#poured the medium　cultured E.coli overnight 1.2 ㎕ into each five microcentritube.
 +
#centrifuged them for 5 min at 5,000 rpm
 +
#prepared new five microcentritube and move 800㎕ the supernatants into each of them.
 +
#measure the OD550 of one tube（use fresh medium as a blank in following assays）
 +
#one hour after, we measured OD 550 of other tube
 +
#take 80㎕ supernatant and move it into new tube and then heated it for 3 min in boiling water and then cooled it in water.
 +
#added 240㎕ DNS reagent and heated it for 3 min in boiling water and then cooled it in water.
 +
#two, three, five hours after, we did above operation, taking supernatant, measured OD500, heating and cooling, applying DNS reagent and heating and cooling again.
 +
#added all sample tube (containing 320㎕ solution) distilled water by 2ｍｌ and measure the absorbance of them in 550nm.
 +
 +
The results of measurement OD550 are shown in fig 3 and of measurement DNS assay are shown in fig 4.

Revision as of 14:00, 5 October 2011

Contents

Measurement

Solubilization of Antibiotics

  1. Mix the following (Final concentration is 50mg/mL).
    • Ampicillin:
      Ampicillin
      1.0g
      MilliQ
      20mL
    • Kanamycin:
      Kanamycin
      0.5g
      MilliQ
      10mL
  2. Dispense 1.1mL of the solution into 1.5mL tubes.
  3. Store in the -20℃ freezer.

Transformation

  1. Unfreeze conpitent cells on ice.
  2. Dry a plate by letting the plate upside down and partly open in incubator.
  3. Add 1µL DNA solution and 20µL compitent cells to 1.5mL tube, let stand for 30min on ice. If few colony is observed, increase the amount of the compitent cells or DNA, but make the amount of DNA not to get over that of the compitent cells.
  4. Heatshock for 60s at 42℃.
  5. Let stand for 2min on ice.
  6. Culture for 1h in preculture medium (LB or SOC medium), and plate by using spreader. Do not heat spreader too much because e.coli will dead for heat.

Sequence

  1. Use Big Dye Terminator 3.1（ABI）
  2. Mix the following
    5xBuffer
    2µL
    Primer (3.2µM)
    1µL
    Template Plasmid 
    200ng
    Big Dye Terminator 3.1
    0.5µL
    MilliQ
    up to 10µL
  3. Let stand for 1min at 96℃.
  4. 35 cycles for 5s at 98℃, for 5s 50℃, and for 2.5min at 68℃.
  5. Add 25µL 100% ethanol and 1µL NAOAC

Measurement of RPU

  1. Cultivate E.coli in M9 media(+ casamino acids) for about 15 hours.
  2. Dispence 2.4ml to each tube.
  3. Centrifuge these tubes (13,000 rpm , 4℃, 1min) and discarded the supernatant.
  4. Add 1.2ml media(- casamino acids) and centrifuged at 4℃ twice.
  5. Centrifuge these tubes again and discarde the supernatant and added 1.2ml media(-casamino acids) at 37 ℃.
  6. Bring up E.coli at 0,5,10,15,20,25,30,60min and extracted RNA and synthesized cDNA.


preliminary experiments

1-1:plotting the standard line


Sample glucose solution(various concentration from 0.20 mM to 1.60 mM)
Blank mixed liquid (240μℓDNS reagent plus 1760㎕distilled water　)


  1. added 240㎕ DNS reagent to 80㎕sample
  2. heated it for 5 min in boiling water and then cooled it in water.
  3. added it distilled water by 2ｍｌ
  4. measured absorbance in 550nm

The standard curve is shown in fig 1.



1-2:evaluate the affection of medium

we examined the interruption based on the components of media(SOC, plas-grow and M9)


Sample each medium
Blank mixed liquid (240μℓDNS reagent plus 1760㎕distilled water　)


  1. added 240㎕ DNS reagent to 80㎕sample
  2. heated it for 5 min in boiling water and then cooled it in water.
  3. added it distilled water by 2ｍｌ
  4. measured absorbance in 550nm

The result is shown in fig 2.

1-3:measurement the effect of remainded E.coli


Sample each medium
Blank mixed liquid (240μℓDNS reagent plus 1760㎕distilled water　)


This assay was performed three times for each medium

  1. poured the medium　cultured E.coli overnight 1.2 ㎕ into each five microcentritube.
  2. centrifuged them for 5 min at 5,000 rpm
  3. prepared new five microcentritube and move 800㎕ the supernatants into each of them.
  4. measure the OD550 of one tube（use fresh medium as a blank in following assays）
  5. one hour after, we measured OD 550 of other tube
  6. take 80㎕ supernatant and move it into new tube and then heated it for 3 min in boiling water and then cooled it in water.
  7. added 240㎕ DNS reagent and heated it for 3 min in boiling water and then cooled it in water.
  8. two, three, five hours after, we did above operation, taking supernatant, measured OD500, heating and cooling, applying DNS reagent and heating and cooling again.
  9. added all sample tube (containing 320㎕ solution) distilled water by 2ｍｌ and measure the absorbance of them in 550nm.

The results of measurement OD550 are shown in fig 3 and of measurement DNS assay are shown in fig 4.

Retrieved from "http://2011.igem.org/Team:Kyoto/Measurement"