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

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

to control the gel extraction

<b> Ligation

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

@@ Line 86: / Line 86: @@
 <li>digestion
 </ul>
 <b>Golden Gate part 2: control gel</b>
@@ Line 595: / Line 594: @@
 </ul>
-<b>Ligation</b>
+<b> Ligation </b>
 <ul>
 <li>of rpd3 into PCR/Blunt

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

Revision as of 21:01, 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