Difference between revisions of "Team:FAU Erlangen/Tour52"

Revision as of 20:32, 18 September 2015

SCATTER

Welcome

Interactive wiki-tour

Menu

Introduction

Planning

Execution

Results

Future

ddH2O: 30.5 µl
dNTP: 4 µl
Phusion-Polymerase: 0.5 µl
Phusion-Buffer (5x): 10 µl
Primer rpd3_fw (5µM): 2.5 µl
Primer rpd3_rv (5µM): 2.5 µl
PCR Program 1 + 2
- 10µl negativ control-sample + 2µl loading dye
- whole PCR tube sample + 8µl loading dye
- 130 V, 45 min
- cut out both 1718bp fragments
- weigh fragments: fragment PCR programm 1: 0.2g fragment PCR programm 2: 0.38g
DNA fragment extraction (protocol Qiagen gel extraction kit)
- eluate extracted DNA in 30µl ddH2O
- control 1%-Agarosegel: 3µl elution + 2µl loading dye
Ligation
- of rpd3 fragment (PCR program 2) into PCR Blunt/StuI
  15/07/01
  
  Transformation
  - Transformation protocol 1 Golden Gate
    - adjust TALE fragments to 10ng/µl (TE buffer) ? mixing ? incubation (50°C, 20') ? mixing
    - each TAL two samples (? 6 samples)
    - TALE
    1. CMV127_1 + CMV127_2 + TALE-CD (= 2416 bp) 2. CMV191_1 + CMV191_2 + TALE-CD (= 2416 bp) 3. GGGT_1 + GGGT_2 + TALE-CD (= 2416 bp)
    - long protocol in ligase buffer
    - sample:
    1x 7x
    T4 ligase buffer (10x) 2 µl 14 µl BSA 2 µl 14 µl T4 Ligase 1 µl 7 µl BsmBI (Esp III) 0.5 µl 3.5 µl 3 TAL fragments each 5.2 µl ddH2O ad 20 µl
    PCR Program 3
    Vector pUC19
    - concentration measurment (NanoDrop): ~ 3µg/µl
    - digestion
    Golden Gate part 2: control gel
    - 10µl TALE sample (1, 2 or 3) + 2µl loading dye
    - 1% Agarose-TAE-Gel
    15/07/02
    
    no white colonies
    
    Transformation
    - Transformation protocol 1 (Repetition rpd3) Repetition pf T4 Ligation
      - rpd3 fragment PCR programm 1 Golden Gate
        
        combine equal samples (6 samples ? 3 samples)
        add 0.5µl BsmBI to each sample, incubate 1h, 55°C
        add 0.5µl Ligase to each sample
        
        PCR Program 4
        
        1% Agarose Gel
        
        15/07/06
        
        Gel extraction
        
        TALEs and pUC19 (protocol Qiagen gel extraction kit) Concentration measurement (NonoDrop)
        pUC19 pUC19 TAL1 TAL2 TAL3 0.9 ng/µl 5.4 ng/µl 2.5 ng/µl 2.0 ng/µl 1.4 ng/µl Ligation
        
        of TALE in pUC19 Fluid culture
        
        12 test tubes: á 2ml LB medium with Kanamycin
        add one white colonie to each tube
        incubation: 300rpm, over night, 37°C
        
        15/07/07
        
        Transformation
        
        Transformation protocol 2 (3x TALE)
        15/07/08
        
        Digestion
        
        of rpd3 Fluid culture
        
        of TALE
        
        6 test tubes: á 2ml LB medium with Kanamycin
        add one white colony to each tube (plate1: 2 colonies; plate2: 3colonies; plate3: 1 colony)
        incubation: 300rpm, over night, 37°C
        
        Gel-electrophorese
        
        15µl digestion probe + 3µl loading dye Sequencing
        
        10µl Klon7 + 20µl ddH2O (M13-RP, GATC IJ1672)
        15/07/09
        
        Golden Gate (2)
        
        1x 4x 7x T4 ligase buffer (10x) 2 µl 8 µl 14 µl BSA 2 µl 8 µl 14 µl T4 Ligase 1 µl 4 µl 7 µl BsmBI (Esp III) 0.5 µl 2 µl 3.5 µl TAL fragments each 5.2 µl ddH2O ad 20 µl PCR Program 5 Miniprep (1)
        
        culture sample ? protocol „Miniscreens“ Digestion
        
        of the mini-prep
        with BamHI and SacI Agarosegel (2)
        
        of the Golden Gate Insert EC culture
        
        3 clones, 2ml LB Ampr Digestion (2)
        
        of the Golden Gate
        15/07/10
        
        Agarosegel (test) (2)
        
        over night digestion golden gate insert 2 Ligation: TALE in Vector pUC19_2
        
        3.5x Mastermix Plurification
        
        of vector 181 (linearazed) Ampr-Leu
        DNA plurification kit ReTrafo
        
        of K801000 iGEM vector Ampr-Ura Miniprep EC insert
        
        Miniscreen protocol, resuspend DNA in 40µl ddH2O Digestion of EC
        22.5 µl DNA 3 µl Tango Buffer —> wrong! 0.5 µl EcoRI 4.5µl µl ddH2O —> 1h @ 37°C —> inactiviation: 20min @ 65°C Sequencing
        
        10µl clone2 + 20µl ddH2O —> mutations
        10µl clone5 + 20µl ddH2O —> mutations Transformation
        
        of Golden Gate (2) Fluid culture
        
        prapare 12 tubes with 2.5ml LB + Amp (4 for each insert) for the weekend
        15/07/13
        
        Digestion
        
        of EC Plasmid-DNA + iGEM vector + 181 vector Miniprep Golden Gate (2)
        
        4 samples of each insert
        protocoll „Miniscreens“
        solve in 40µl ddH2O Digestion
        
        of Golden Gate (2) Agarose gel
        
        of EC + 181 + iGEM vector
        + 5µl Loading Dye
        complete digestion (30µl) Plurification
        
        EC1 + 2 —> plurification kit Ligation
        
        EC2 ligation into 181 and iGEM Agarose gel
        
        2µl Golden Gate digestion sample + 0.5µl Loading Dye Sequencing
        
        Insert 2_2: 3µl + 17µl ddH2O Golden Gate (3)
        1x 4x T4 ligase buffer (10x) 2 µl 8 µl BSA 2 µl 8 µl T4 Ligase 1 µl 4 µl BsmBI (Esp III) 0.5 µl 2 µl TAL fragments each 5.2 µl ddH2O ad 20 µl PCR Program 5
        15/07/14
        
        Addition of ligase (3)
        
        because golden gate was not optimal 1x T4 ligase buffer (10x; new) 2 µl T4 Ligase 1 µl BsmBI (Esp III) 0.5 µl Golden Gate reaction; each 10 µl ddH2O 8.5 µl —> 40' @ room temperature Digestion (3)
        
        of Golden Gate-reaction
        with BamHI and SacI
        Inactivation of the enzymes for 20 min at 80°C Transformation
        
        of the ligation Agarose gel
        
        of the digested Golden Gate reaction
        1/5 of the digestion with 1.5 µl Loading Dye Sequencing
        
        Clone 10 + Clone 11 of rpd3 Fluid culture
        
        one white colony of the ReTrafo of the iGEM vector in 20ml LB-medium Ligation (3)
        
        of the Golden Gate reaction into pUC19
        15/07/15
        
        Transformation
        
        of the Golden Gate ligation Mixi-Prep
        
        of the iGEM vector
        protocol „mixi-prep“ Ligation
        
        of EC2-fragment into 181-vector
        because there were no white colonies last time, we do a new ligation Fluid culture
        
        of 6 clones of EC2 + iGEM-vector Transformation
        
        of the rpd3-ligation
        15/07/16
        
        Mini-Prep
        
        of EC + iGEM-vector
        protocol „miniscreens“ Transformation
        
        of EC2 + 181-vector ligation (of 15/07/07)
        and Golden Gate ligation (of 15/07/14) Digestion
        
        of the mini-prep Plating
        
        of the transformation onto LB-plates containing ampicillin, XGal and IPTG Agarose gel
        
        of the digestion
        add 4µl of Loading Dye
        15/07/17
        
        Digestion
        
        of the Golden Gate reaction (of 15/07/13), because no colonies grew
        first incubate 1h at 37°C with only SacI
        then add BamHI and incubate for another hour Digestion
        
        of the 181-vector, because there were only blue colonies on the plates Fluid culture
        
        of rpd3 —> 12 clones
        and of 2 blue clones of EC + 181 to test them Ligation
        
        of Golden Gate into pUC19
        of EC into 181-vector
        15/07/20
        
        Mini-prep
        
        of the fluid cultures from 15/07/17
        protocol „miniscreens“ Transformation
        
        of the ligations from 15/07/17 Digestion
        
        of the mini-preps Agarose gel
        
        of the digestion
        rpd3: 15µl with 3µl loading dye
        EC/181: 25µl with 5µl loading dye Sequencing
        
        of 2 rpd3 clones
        15/07/22
        
        Fluid culture
        
        of 4 clones for Golden Gate
        and 4 clones of EC + 181
        15/07/23
        
        Mini-prep
        
        of the fluid cultures
        protocol „miniscreens“ Digestion
        
        of the mini-preps Agarose Gel
        
        of the digestion
        with 3µl loading dye Overlap-PCR
        
        of rpd3
        PCR program of 15/06/30 Sequencing
        
        of Golden Gate fragment 1 and 3
        15/07/24
        
        Agarose gel
        
        of the overlap-PCR Gel extraction
        
        with Qiagen Gel Extraction Kit Agarose gel
        
        to control the gel extraction <b>Ligation</b>
        
        of rpd3 into PCR/Blunt
        protocol of 15/06/30

Next chapter

Next chapter

Dig deeper

End of tour
Want more? Go to iGEM.org!

If you want to learn more about Synthetic Biology you should visit iGEM's homepage, which contains loads on the subject. You can even visit other teams' wikis and take a look at what they accomplished. Or maybe join a team yourself?

A tour for everyone
Click here to begin the interactive wiki-tour

Take a quick glance at our project or dig deeper to get a complete walkthrough of the wiki. Or do something in-between, the choice is yours.

@@ Line 20: / Line 20: @@
 <li>Primer rpd3_fw (5µM): 2.5 µl
 <li>Primer rpd3_rv (5µM): 2.5 µl
+</ul>
 <p>negative control:</p>

Difference between revisions of "Team:FAU Erlangen/Tour52"

Revision as of 20:32, 18 September 2015

Contents

15/06/30

15/07/01

15/07/02

15/07/06

15/07/07

15/07/08

15/07/09

15/07/10

15/07/13

15/07/14

15/07/15

15/07/16

15/07/17

15/07/20

15/07/22

15/07/23

15/07/24