User:DrJones1935/22 July 2012

From 2012.igem.org

(Difference between revisions)
(Created page with "====I. PCR==== *'''Mix Reagents:''' :*Mix according to the following table, where 2 = LacZ, 3 = CAT*, 4 = tet<sup>r</sup>, 0.1-0.3 = +template, and 0.4 = -template ::*For exampl...")
(→I. PCR)
 
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====I. PCR====
+
====I. Re-run AGE on PCR samples====
 +
 
 +
*''SAME RESULTS AS ABOVE''
 +
 
 +
====II. Re-run PCR of questionable samples====
 +
 
*'''Mix Reagents:'''
*'''Mix Reagents:'''
-
:*Mix according to the following table, where 2 = LacZ, 3 = CAT*, 4 = tet<sup>r</sup>, 0.1-0.3 = +template, and 0.4 = -template
+
:*Mix according to the following table, where 2 = LacZ, 3 = CAT*, 0.1-0.3 = +template, and 0.4 = -template
::*For example, 2.1 = lacZ sample, 2.4 = lacZ control
::*For example, 2.1 = lacZ sample, 2.4 = lacZ control
{| class="wikitable"
{| class="wikitable"
|-
|-
-
!  !! 2.1 !! 2.2 !! 2.3 !! 2.4 !! 3.1 !! 3.2 !! 3.3 !! 3.4 !! 4.1 !! 4.2 !! 4.3 !! 4.4
+
!  !! 2.1 !! 2.2 !! 2.3 !! 2.4 !! 3.1 !! 3.2 !! 3.3 !! 3.4  
|-
|-
-
! Reagent !! (uL) !! (uL) !! (uL) !! (uL) !! (uL) !! (uL) !! (uL) !! (uL) !! (uL) !! (uL) !! (uL) !! (uL)
+
! Reagent !! (uL) !! (uL) !! (uL) !! (uL) !! (uL) !! (uL) !! (uL) !! (uL)
|-
|-
-
| water || 33.5 || 33.5 || 33.5 || 33.5 || 33.5 || 33.5 || 33.5 || 33.5 || 32.5 || 32.5 || 32.5 || 33.5
+
| water || 33.5 || 33.5 || 33.5 || 33.5 || 33.5 || 33.5 || 33.5 || 33.5  
|-
|-
-
| 5x Phusion HiFi buffer || 10 || 10 || 10 || 10 || 10 || 10 || 10 || 10 || 10 || 10 || 10 || 10
+
| 5x Phusion HiFi buffer || 10 || 10 || 10 || 10 || 10 || 10 || 10 || 10
|-
|-
-
| 10 mM dNTP mix || 1 || 1 || 1 || 1 || 1 || 1 || 1 || 1 || 1 || 1 || 1 || 1
+
| 10 mM dNTP mix || 1 || 1 || 1 || 1 || 1 || 1 || 1 || 1  
|-
|-
-
| 10 uM primer (F) || 2.5 (p2f_2) || 2.5 (p2f_2 || 2.5 (p2f_2) || 2.5 (p2f_2) || 2.5 (p3f_2) || 2.5 (p3f_2) || 2.5 (p3f_2) || 2.5 (p3f_2) || 2.5 (p4f) || 2.5 (p4f) || 2.5 (p4f) || 2.5 (p4f)
+
| 10 uM primer (F) || 2.5 (p2f_2) || 2.5 (p2f_2 || 2.5 (p2f_2) || 2.5 (p2f_2) || 2.5 (p3f_2) || 2.5 (p3f_2) || 2.5 (p3f_2) || 2.5 (p3f_2)  
|-
|-
-
| 10 uM primer (R) || 2.5 (p2r_2) || 2.5 (p2r_2) || 2.5 (p2r_2) || 2.5 (p2r_2) || 2.5 (p3r) || 2.5 (p3r) || 2.5 (p3r) || 2.5 (p3r) || 2.5 (p4r_2) || 2.5 (p4r_2) || 2.5 (p4r_2) || 2.5 (p4r_2)
+
| 10 uM primer (R) || 2.5 (p2r_2) || 2.5 (p2r_2) || 2.5 (p2r_2) || 2.5 (p2r_2) || 2.5 (p3r) || 2.5 (p3r) || 2.5 (p3r) || 2.5 (p3r)  
|-
|-
-
| Template || ECNR2 colony* || ECNR2 colony* || ECNR2 colony* || 0 || ECFI5 colony* || ECFI5 colony* || ECFI5 colony* || 0 || 1 (pBR322) || 1 (pBR322) || 1 (pBR322) || 0  
+
| Template || ECNR2 colony* || ECNR2 colony* || ECNR2 colony* || 0 || ECFI5 colony* || ECFI5 colony* || ECFI5 colony* || 0  
|-
|-
-
| Phusion HiFi polymerase || 0.5 || 0.5 || 0.5 || 0.5 || 0.5 || 0.5 || 0.5 || 0.5 || 0.5 || 0.5 || 0.5 || 0.5
+
| Phusion HiFi polymerase || 0.5 || 0.5 || 0.5 || 0.5 || 0.5 || 0.5 || 0.5 || 0.5
|}
|}
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|}
|}
*Store tubes on ice after PCR is finished
*Store tubes on ice after PCR is finished
 +
 +
====III. Agarose Gel Electrophoresis (AGE) of PCR Samples====
 +
 +
*'''Make Gel:'''
 +
:*Measure out <strike>0.5 g</strike> 0.50231 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 <sup>o</sup>C 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:
 +
{| class="wikitable"
 +
|-
 +
! Ladder (Invtrogen 1 kb plus) !! Blank !! Sample
 +
|-
 +
| *5 uL DNA ladder<br>*1 uL 6x Loading Dye || *8.3 uL dH<sub>2</sub>O<br>*1.7 uL 6x Loading Dye || *6.3 uL dH<sub>2</sub>O<br>*1.7 uL 6x Loading Dye<br>*2.0 uL DNA
 +
|}
 +
:*Load ~6 uL on gel according to the following chart:
 +
{| class="wikitable"
 +
|-
 +
! Lane 1 !! 2 !! 3 !! 4 !! 5 !! 6 !! 7 !! 8 !! 9 !! 10 !! 11
 +
|-
 +
| NEB 2-log ladder || Blank || 2.1 || 2.2 || 2.3 || 2.4 || 3.1 || 3.2 || 3.3 || 3.4
 +
|}
 +
:*Store DNA at 4 <sup>o</sup>C
 +
 +
*'''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 the well
 +
 +
*'''Image Gel:'''
 +
:*''RESULTS:''
 +
[[File:Srk_2012-07-22_19hr_22min.jpg|center|500px]]
 +
Source: [[Media:Srk_2012-07-22_19hr_22min.tif]]
 +
{| class="wikitable"
 +
|-
 +
! Feature !! Expected Size (bp)
 +
|-
 +
| LacZ || 3151
 +
|-
 +
| CAT* || 729
 +
|}
 +
<br>
 +
<br>
 +
 +
----
====II. Agarose Gel Electrophoresis (AGE) of PCR Samples====
====II. Agarose Gel Electrophoresis (AGE) of PCR Samples====

Latest revision as of 23:25, 3 October 2012

Contents

I. Re-run AGE on PCR samples

  • SAME RESULTS AS ABOVE

II. Re-run PCR of questionable samples

  • Mix Reagents:
  • Mix according to the following table, where 2 = LacZ, 3 = CAT*, 0.1-0.3 = +template, and 0.4 = -template
  • For example, 2.1 = lacZ sample, 2.4 = lacZ control
2.1 2.2 2.3 2.4 3.1 3.2 3.3 3.4
Reagent (uL) (uL) (uL) (uL) (uL) (uL) (uL) (uL)
water 33.5 33.5 33.5 33.5 33.5 33.5 33.5 33.5
5x Phusion HiFi buffer 10 10 10 10 10 10 10 10
10 mM dNTP mix 1 1 1 1 1 1 1 1
10 uM primer (F) 2.5 (p2f_2) 2.5 (p2f_2 2.5 (p2f_2) 2.5 (p2f_2) 2.5 (p3f_2) 2.5 (p3f_2) 2.5 (p3f_2) 2.5 (p3f_2)
10 uM primer (R) 2.5 (p2r_2) 2.5 (p2r_2) 2.5 (p2r_2) 2.5 (p2r_2) 2.5 (p3r) 2.5 (p3r) 2.5 (p3r) 2.5 (p3r)
Template ECNR2 colony* ECNR2 colony* ECNR2 colony* 0 ECFI5 colony* ECFI5 colony* ECFI5 colony* 0
Phusion HiFi polymerase 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

*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

III. Agarose Gel Electrophoresis (AGE) of PCR Samples

  • Make Gel:
  • Measure out 0.5 g 0.50231 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 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
*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 ~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 Blank 2.1 2.2 2.3 2.4 3.1 3.2 3.3 3.4
  • 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 ~halfway down the well
  • Image Gel:
  • RESULTS:
Srk 2012-07-22 19hr 22min.jpg

Source: Media:Srk_2012-07-22_19hr_22min.tif

Feature Expected Size (bp)
LacZ 3151
CAT* 729



II. Agarose Gel Electrophoresis (AGE) of PCR Samples

  • Make Gel:
  • Measure out 0.5 g 0.50238 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 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
*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 ~10 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 12 13 14 15
NEB 2-log ladder Blank 2.1 2.2 2.3 2.4 3.1 3.2 3.3 3.4 4.1 4.2 4.3 4.4 NEB 2-log ladder
  • 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 near the bottom of the gel
  • Image Gel:
  • RESULTS:
Srk 2012-07-21 17hr 37min.jpg

Source: Media:Srk_2012-07-21_17hr_37min.tif

Feature Expected Size (bp)
Leftover pBR322 4361
LacZ 3151
CAT* 729
tetr 1265



Retrieved from "http://2012.igem.org/User:DrJones1935/22_July_2012"