Difference between revisions of "Team:FAU Erlangen/Tour52"
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<li>Transformation protocol 1 | <li>Transformation protocol 1 | ||
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<b>Golden Gate</b> | <b>Golden Gate</b> | ||
Revision as of 20:38, 18 September 2015
Contents
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:
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
- 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
- to control the gel extraction
<b>Ligation</b>
- with Qiagen Gel Extraction Kit
Agarose gel
- of the overlap-PCR
Gel extraction
- of Golden Gate fragment 1 and 3
- of the mini-preps
Agarose Gel
- of 2 rpd3 clones
- of the mini-preps
Agarose gel
- of the ligations from 15/07/17
Digestion
- of the 181-vector, because there were only blue colonies on the plates
Fluid culture
- of the transformation onto LB-plates containing ampicillin, XGal and IPTG
Agarose gel
- of the mini-prep
Plating
- of the rpd3-ligation
- of 6 clones of EC2 + iGEM-vector
Transformation
- of the Golden Gate ligation
Mixi-Prep
- of the Golden Gate reaction into pUC19
- one white colony of the ReTrafo of the iGEM vector in 20ml LB-medium
Ligation (3)
- Clone 10 + Clone 11 of rpd3
Fluid culture
- of the ligation
Agarose gel
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)
- Insert 2_2: 3µl + 17µl ddH2O
Golden Gate (3)
- 2µl Golden Gate digestion sample + 0.5µl Loading Dye
Sequencing
- EC2 ligation into 181 and iGEM
Agarose gel
- EC1 + 2 —> plurification kit
Ligation
- of Golden Gate (2)
Agarose gel
- of EC Plasmid-DNA + iGEM vector + 181 vector
Miniprep Golden Gate (2)
- prapare 12 tubes with 2.5ml LB + Amp (4 for each insert) for the weekend
- of Golden Gate (2)
Fluid culture
- Miniscreen protocol, resuspend DNA in 40µl ddH2O
Digestion of EC
- of K801000 iGEM vector Ampr-Ura
Miniprep EC insert
- 3.5x Mastermix
Plurification
- over night digestion golden gate insert 2
Ligation: TALE in Vector pUC19_2
- of the Golden Gate
- 3 clones, 2ml LB Ampr
Digestion (2)
- of the Golden Gate
Insert EC culture
- culture sample ? protocol „Miniscreens“
Digestion
- 10µl Klon7 + 20µl ddH2O (M13-RP, GATC IJ1672)
- of TALE
- of rpd3
Fluid culture
- of TALE in pUC19
Fluid culture
- rpd3 fragment PCR programm 1
Golden Gate