Difference between revisions of "Team:Paris Bettencourt/Notebook/Riboswitch"

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L2 ++ (50) <br>
 
L2 ++ (50) <br>
 
L3 +++ (100)<br>
 
L3 +++ (100)<br>
 +
 +
<br><br>
 +
<h1 class="date two">August 12th</h1>
 +
 +
<h2>Goal 1 </h2>
 +
Ligation of LP2 alone and transfo for a negative control
 +
 +
<h2>Procedure</h2>
 +
Vector : LP2 (40.1ng/uL) 2 uL<br>
 +
No insert <br>
 +
T4 ligase buffer : 4 uL <br>
 +
T4 ligase : 2 uL <br>
 +
Water : 32 uL <br>
 +
TOTAL : 40 uL <br>
 +
Transfo : use 3 uL of ligated product <br>
 +
Result after culture on LB + Cm : nothing for the C and lot of colonies for L2 <br>
 +
 +
<h2>Goal 2 </h2>
 +
Colony PCR on yesterday transformations plates
 +
 +
<h2>Procedure</h2>
 +
Using primers 148 - 149 (10uM), 2 uL each<br>
 +
Dream Taq Master Mix : 10 uL<br>
 +
Water : 6uL<br>
 +
TOTAL : 20uL<br>
 +
<style type="text/css">
 +
.tg  {border-collapse:collapse;border-spacing:0;}
 +
.tg td{font-family:Arial, sans-serif;font-size:14px;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;}
 +
.tg th{font-family:Arial, sans-serif;font-size:14px;font-weight:normal;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;}
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</style>
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<table class="tg">
 +
  <tr>
 +
    <th class="tg-031e">Colony n°</th>
 +
    <th class="tg-031e">1</th>
 +
    <th class="tg-031e">2</th>
 +
    <th class="tg-031e">3</th>
 +
    <th class="tg-031e">4</th>
 +
    <th class="tg-031e">5</th>
 +
    <th class="tg-031e">6</th>
 +
    <th class="tg-031e">7</th>
 +
    <th class="tg-031e">8</th>
 +
    <th class="tg-031e">9</th>
 +
    <th class="tg-031e">10</th>
 +
    <th class="tg-031e">11</th>
 +
    <th class="tg-031e">12</th>
 +
    <th class="tg-031e">13</th>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-031e">Comes from</td>
 +
    <td class="tg-031e">L2-1</td>
 +
    <td class="tg-031e">L2-2</td>
 +
    <td class="tg-031e">L2-3</td>
 +
    <td class="tg-031e">L2-4</td>
 +
    <td class="tg-031e">L2-5</td>
 +
    <td class="tg-031e">L1-1</td>
 +
    <td class="tg-031e">L1-2</td>
 +
    <td class="tg-031e">L1-3</td>
 +
    <td class="tg-031e">L1-4</td>
 +
    <td class="tg-031e">L3-1</td>
 +
    <td class="tg-031e">L3-2</td>
 +
    <td class="tg-031e">L3-3</td>
 +
    <td class="tg-031e">L3-4</td>
 +
  </tr>
 +
</table>

Revision as of 18:18, 14 September 2015

July 27th

Goal

E coli DH5-alpha transformation with RFP from iGEM plate 4 - 2H (pSB1A3 backbone)

Procedure

  1. I took iGEM plate 4 - 2H (RFP, BBa_J04450 in PSB1A3 backbone) and resuspend it in 10 uL of distilled water.
  2. Used E coli competent cells from NEB 2 tubes c2987 (50uL) : 1 control (C) and one for transformation (T)
  3. Introduced 2 uL (200-300pg/ul) of my suspended plasmid solution in T.
  4. Waiting 30 min in ice
  5. Heat shock 30 sec 42°C
  6. Then 5min in ice
  7. Add 200uL SOC medium
  8. Recovery 2 hours then plate it on 3 different 100ug/mL Ampicillin LB
  9. Incubate 37°C till tomorrow

Results

The day after I had colonies growing, nothing on my control
    I took 2 different colonies and do a 8 hours culture in two different tubes T1 an T2 in LB + Amp 100ug/mL, in order to do a glycerol stock.


July 29th

Goal

Making chemically competent cells

Procedure

I followed the following method
  • The day before, start an overnight Liquid Culture of the E. coli strain. (5mL of LB + Antibiotics + Single colony)
  • The next day, set the Centrifuge at 4°C and place on ice a tube containing 15mL of 100mM CaCl2 and a tube containing 4mL of 100mM CaCl2 + 15% Glycerol.
  • In an Erlenmeyer, add 50mL of LB and 250uL of the Liquid Culture, (for quicker growth, use a larger Erlenmeyer)
  • Put the Erlenmeyer to shake at 37°C until the OD(600) reaches 0.4 - 0.5.
  • When the OD(600) is reached, transfer in a 50mL Falcon tube and centrifuge 10min at 4°C.
  • Remove the supernatant and resuspend in 15mL of ice cold 100mM CaCl2. -Leave on ice for 4h.
  • Centrifuge 10min at 4°C.
  • Remove the supernatant and resuspend in 4mL of ice cold 100mM CaCl2 + 15% Glycerol.
  • Aliquot in 1.5mL eppendorf tubes and store at -80°C until use.


  • July 30th

    Goal 1

    PCR my G block and backbone

    Procedure

  • PCR1 = Backbone + 068 and 069 primers, before purify
  • PCR2 = FINAL + 068 and 069 primers, before purify
  • Then PCR purification
  • Nanodrop : PCR 1 = 61ng/uL ; PCR2 = 25,4 ng/uL
  • Goal 2

    Digestion/Ligation/Transformation

    Procedure

    First, digestion :
  • PCR1 ADN : 40uL = 2,4 ug 4uL EcoRI, PstI, FastAp 12 uL FD buffer 56 uL water TOT =120uL
  • PCR2 ADN : 40uL = 1ug 6 uL EcoRI, PstI 12 uL FD buffer 64 uL water TOT= 128uL
  • 10 min at 37°C

  • Then PCR purification
  • Nanodrop : nanodrop : PCR 1 = 20,7 ng/uL ;PCR2 = 25,7

  • For the ligation :
  • Vector PCR1 100 ng = 5 uL; Insert PCR 2 300 ng = 12uL T4 ligase buffer 2uL ; T4 ligase 1 uL ; Total 20 uL
  • 20-40 min on the bench

  • Nanodrop : L= 270 ng/uL

  • Then I did a heat shock transformation (following the iGEM protocol : http://parts.igem.org/Help:Protocols/Transformation )
  • C + = 10 ng/uL of RFP in pS1C3, 2 uL transformed (10pg/ul RFP Control (pSB1C3 w/ BBa_J04450)
  • C - = nothing but efficients cells
  • T = Plasmid construct 2uL transformed
  • Each with 50 uL of chemically competents cells done yesterday.

  • Then I did 5 different plates :
  • 5 plates with 25ug/mL chloramphenicol in LB
  • 200 uL control + , 20 uL control +
  • 200 uL T , 20 uL T
  • 200 uL C -
  • Incubation overnight at 37°C


  • July 31th

    Goal 1

    Check the transformations I've done yesterday

    Procedure

    According to my plates my competent cells are working, red colonies on the C+. Transformation of my construction didn’t worked, I’ll start again all the process : PCR, purify, digest, ligate, integrate (heat shock), overnight culture.

    Goal 2

    PCR my backbone and my insert

    Procedure

  • PCR1' = Backbone + 068 and 069 primers, before purify
  • PCR2' = FINAL + 068 and 069 primers, before purify
  • After PCR and after purification : 1'p = 66,5 2'p =90,5 ng/uL
  • The gel migration shows a bad result for PCR 1’ (Linearized pSB1C3)


  • August 1st

    Goal

    PCR my backbone and my insert again : 1" and 2"

    Procedure

    PCR then purify

    August 5th

    Goal

    Ligation/transformations (insertion of FINAL block from IDT in the pSB1C3 backbone, FINAL is my Riboswitch associated with an EGFP)

    Procedure

  • Used 3 different insert PCR products
  • Ligation after digestion and purification of PCR2’ = 94,6ng/uL , PCR2’’= 57,5 ng/uL , PCR2’’’=83,4 ng/uL ; Backbone pSB1C3 = 37,9ng/uL
  • ' " "'
    Insert 4 7 5
    Backbone 5 5 5
    Buffer 2 2 2
    Ligase 1 1 1
    Water 8 5 7
    TOTAL 20uL 20uL 20uL
    I did then a transformation using the iGEM protocol (Heat Shock) with 50uL of chemically competent cells + 3 uL of Ligation Product for each (pSB1C3-TOTAL)
  • C + : pSB1C3-RFP
  • C - : just competent cells

  • Plated on Cm 25 ug/mL
  • 5 plates : ‘,’’,’’’,C+,C-
  • Results

  • C- : nothing
  • C+ : lot of red colonies
  • ‘ : lot
  • ‘’ : lot
  • ‘’’ : lot
  • I should have done a control transformation with just the backbone


  • August 6th

    Goal 1

    Cultivate my transformants

    Procedure

  • There is red and white transformant colonies, all resistant to Cm 25
  • I selected some white colonies overnight culture
  • On plate LB Cm —> it grew (some red, maybe contamination)
  • On broth LB Cm —> it grew (some red, maybe contamination)
  • Inoculated at 4:00 pm in LB Cm 25 bacteria from a culture ( red —> RFP ) with different AdoCbl concentrations in order to detect the minimal amount of B12 viable for e.coli : 5; 10; 20; 40; 80; 160 ng/mL
  • Goal 2

    PCR the pSB1C3 backbone and my FINAL riboswitch construction

    Procedure

  • 2 PCR each
  • I1 and I2 using primers 150 and 151
  • LP1 and LP2 using primers 152 and 153
  • Purification then nano drop : good for I2 : 112.4 ng/uL
  • Results

    It shows a good length only for I2 at 1300 bp.

    Goal 3

    Do the PCR again

    Procedure

    Template Primers MasterMix Water TOTAL Annealing T°C Elongation time
    I3 2ng = 2 uL 1 uL 25 uL 22 uL 50uL 56°C 2 min
    I4 2ng = 2 uL 2 uL 25 uL 21 uL 50uL
    LP3 25ng = 1uL 1 uL 25 uL 23 uL 50uL
    LP4 25ng = 1uL 2 uL 25 uL 22 uL 50uL

    Results

    After PCR purify, nanodrop (ng/uL)
    I3 I4 LP3 LP4
    30.7 177.6 86.7 93.9
    Gel migration
    Very good for LP4, good for LP3
    Nothing for I3 and I4


    August 7th

    Goal 1

    Digestion of LP4 and I2

    Procedure

    LP4 I2
    EcoRI 4uL 4uL
    PstI 4uL 4uL
    FastAP 4uL -
    FD Buffer 12uL 8uL
    DNA 2 ug (90 ng/uL) —> 20 uL 2ug (100 ng/uL) —> 20 uL
    Water 76 uL 44 uL
    TOTAL 120uL 80uL
    New nano drop : LP4 = 87,4 ; I2 = 90,4 ng/uL
    Waited 15 min at 37°C
    After digestion and PCR purification, nanodrop :
    38.2 ng/uL for I2 (d. and p.)
    31.2 ng/uL for LP4 (d. and p.)

    Goal 2

    Ligation of LP4dp and I2dp

    Procedure

    L*
    Vector LP4 dp 100 ng (31.2 ng/uL) —> 3uL
    Insert I2 dp 300 ng (38.2 ng/uL) —> 8uL
    T4 L,Buffer 2uL
    T4 ligase 1uL
    Water 6uL
    TOTAL 20 uL
    Waited 40 min on the bench (25°C)

    Goal 3

    Transformation of L*

    Procedure

    C- C+ T*
    ChemicallyCompetent Cells 50 uL 50 uL 50 uL
    L* - - 3 uL
    pSB1C3 with RFP 100ng/uL - 3 uL -
    iGEM Transfo protocol
    90 sec heat shock
    On saturday they were growing but no fluorescence

    August 10th

    Goal 1

    Colony PCR my T*

    Procedure

    Pick up 16 colonies on my two T* plates
    Put some bacteria in PCR tube + 2 uL of 148 and 149 (iGEM verification forward and backward) ;
    10uM + 50 uL of Master Mix Phusion (last in the tubes) + 45 uL of water (first in the tube)
    Waiting for a 1577 bp fragment on a gel Gel migration with a GR 1kb

    It is quiet bad

    August 11th

    Goal 1

    Do a new PCR of FINAL (G-block) and of pSB1C3 associated with RFP

    Procedure

    Template Primers Master Mix Phusion Water Time of elongation (72°C) T°C annealing Lenght Primers Nanodrop after purify (ng/uL)
    LP1* 5ng 2uL 25uL QSP for 50uL 2 min 56°C 2078 152 - 153 68.3
    LP2* 10ng 2uL 25uL QSP for 50uL 2 min 56°C 2078 152 - 153 133.5
    LP3* 2ng 2uL 25uL QSP for 50uL 2 min 56°C 2078 152 - 153 127.8
    F1* 2ng 2uL 25uL QSP for 50uL 1 min 58°C 1314 150 - 151 98
    F2* 3ng 2uL 25uL QSP for 50uL 1 min 58°C 1314 150 - 151 102
    F3* 1ng 2uL 25uL QSP for 50uL 1 min 58°C 1314 150 - 151 58

    Results


    Gel migration wit 1kb GR

    Goal 2

    Digestion of pSB1C3 + RFP

    Procedure

    Backbone+RFP
    Plasmid (100ng/uL) 10uL
    EcoRI 2uL
    PstI 2uL
    Buffer 6uL
    Water 38uL
    FastAP 2uL
    TOTAL 60uL

    Results

    Gel migration : order = 1kb GR - Digested product x3

    Then I cuted the 2100 fragment and did the gel extraction protocol

    Goal 3

    Digestion of pSB1C3 linearized (LP1,2,3 *) and F1,2,3 *

    Procedure

    F2d LP2d LP3d
    ADN 10 uL 10 uL 10 uL
    EcorRI 2 uL 2 uL 2 uL
    PstI 2 uL 2 uL 2 uL
    Buffer 4 uL 6 uL 6 uL
    Water 22 uL 38 uL 38 uL
    FastAP _ 2uL 2uL
    TOTAL 40 60 60
    Then 30min at 37°C

    Goal 4

    Ligations and transformations

    Procedure

    L1 L2 L3
    Vector 100ng LP2d (40.1ng/uL),2uL LP3d (21.1ng/uL),4 uL Backbone from digestion + electrophoresis extraction (9.1ng/uL), 10 uL
    Insert 300 ng (8.1ng/uL) (F2d) 30 uL 20 uL 10 uL
    T4 ligase buffer 4 uL 4 uL 2 uL
    T4 ligase 2 uL 2 uL 1 uL
    Water 0 10 uL 0
    TOTAL 40 uL 40 uL 20 uL
    Waited 20 min on the bench after mixing to do the ligation
    Used 3 uL for the transformations with 50 uL of competent cells
    (iGEM protocol) Waited 15 min instead of 30 min after put the ligation product (on ice)
    Heat shock 1 min 30
    2 hours recovery
    Plated C- (just chemically competents cells Heat Shocked) C (LP2 dp ! no ligation) L1, L2, L3

    Results

    Grew on L1, L2, L3 ; nothing on the two others
    Number of colonies :
    L1 ++ (50)
    L2 ++ (50)
    L3 +++ (100)


    August 12th

    Goal 1

    Ligation of LP2 alone and transfo for a negative control

    Procedure

    Vector : LP2 (40.1ng/uL) 2 uL
    No insert
    T4 ligase buffer : 4 uL
    T4 ligase : 2 uL
    Water : 32 uL
    TOTAL : 40 uL
    Transfo : use 3 uL of ligated product
    Result after culture on LB + Cm : nothing for the C and lot of colonies for L2

    Goal 2

    Colony PCR on yesterday transformations plates

    Procedure

    Using primers 148 - 149 (10uM), 2 uL each
    Dream Taq Master Mix : 10 uL
    Water : 6uL
    TOTAL : 20uL
    Colony n° 1 2 3 4 5 6 7 8 9 10 11 12 13
    Comes from L2-1 L2-2 L2-3 L2-4 L2-5 L1-1 L1-2 L1-3 L1-4 L3-1 L3-2 L3-3 L3-4