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

15/06/30

Primer/rpd3 sequence fragments:

• centrifugation (5'; 13,000rpm): RPD3 sequences and primer
• adjust forward and reverse primer to 100µM (ddH2O) ? working concentration: 5µM (10µl primer + 190µl ddH2O)
• adjust RPD3 fragments to 10ng/µl (TE buffer) ? mixing ? incubation (50°C, 20') ? mixing

PCR

• ddH2O: 28.5 µl
• rdp3 fragment 2.1: 1 µl
• rdp3 fragment 2.2: 1 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

negative control:

• 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:
• T4 ligase buffer (10x): 2 µl
• BSA: 2 µl
• T4 Ligase: 1 µl
• BsmBI (Esp III): 0.5 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: 0.9 ng/µl
• pUC19: 5.4 ng/µl
• TAL1: 2.5 ngµl
• TAL2: 2.0 ng/µl
• TAL3: 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)

• T4 ligase buffer (10x): 2 µl
• BSA: 2 µl
• T4 Ligase: 1 µl
• BsmBI (Esp III): 0.5 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
• 0.5 µl EcoRI
• 4.5µl µl ddH2O
• 1h at 37°C
• inactiviation: 20min at 65°C

Sequencing

• 10µl clone2 + 20µl ddH2O —> mutations
• 10µl clone5 + 20µl ddH2O —> mutations

Transformation

• of Golden Gate (2)

Fluid culture

• prepare 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)

• T4 ligase buffer (10x): 2 µl
• BSA: 2 µl
• T4 Ligase: 1 µl
• BsmBI (Esp III): 0.5 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
• 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' at 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

<b>Ligation

• of rpd3 into PCR/Blunt
• protocol of 15/06/30