Team:Goettingen/NoteBook w13

From 2013.igem.org

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Revision as of 13:59, 24 September 2013

August

27th

Test gel for DarR_rev-Term_rev C3 vector after 2nd digest (with PstI) and dephosphorylation, Plasmid Mini Preparation, Test gel for plasmids after Plasmid Mini Preparation and part 6.4 A/B vectors....

Test gel for DarRrev-Termrev C3 vector after 2nd digest (with PstI)

- 1 % agarose-1x TAE gel

- loading of 3 μl 2 log ladder

- loading of 3 μl RD reaction + 1 μl dH2O + 1 μl 5xLD

- loading of 1 μl uncut plasmid + 3 μl dH2O + 1 μl 5xLD

- rut at 100 V

- EtBr staining + destaining in water

- UV detection

loading: Marker/uncut DarRrev-Termrev C3 plasmid/ RD DarRrev-Termrev C3 vector

digest still partial (expected band at ca. 3kb is observed, but also a weak band at ca. 6 – 8 kb of uncut plasmid) → dephosphorylation of vector with AP to avoid self-ligation

Preparation of LB media

- 1x 300 ml broth LB w/o antibiotics

- 1x 500 ml LBCm plates

Dephosphorylation of DarRrev-Termrev C3 vector cut with SpeI and PstI

ca. 37 μl digest reaction

+ 2 μl AP

+ 5 μl 10x AP buffer

+ 6 μl dH2O

incubation for 1.5 h at 37 °C (PstI forms 3’ overhang, but dephosphorylation often incomplete → incubation for more than 1 h)

Preparation of CryoStocks of clones inoculated yesterday

DMSO cryostocks prepared as described before (13.08.2013)

for all clones inoculated on 26.8.13:

- part 1 C2

- part 2 C3

- part 3 C2 and C3

- part 4 C2 and C3

- DarRrev-Termrev in pSB1C3 C3

- part 6.4 A C3

- part 6.4 B C2 and C5

- Promoter1rev in pSB1C3 C4

- Promoter3rev in pSB1C3 C5

Plasmid Mini Preparation

- harvesting of all cultures inoculated yesterday

- but MiniPrep only for the following clones:

DarRrev-Termrev in pSB1C3 C3

part 6.4 A C3

part 6.4 B C2 and C5

Promoter1rev in pSB1C3 C4

Promoter3rev in pSB1C3 C5

- all other clones were harvested and the cell pellets stored in to-do-box (it could be that we won’t need the plasmids…):

part 1 C2

part 2 C3

part 3 C2 and C3

part 4 C2 and C3

- elution 1x with 30 μl HPLC water (pre-warmed), incubation at 50 °C for 2 min

- NanoDrop concentration measurement:

Test gel for plasmids after Plasmid Mini Preparation and part 6.4 A/B vectors

- 1 % agarose-1x TAE gel

- loading of 3 μl 2 log ladder

- loading of 3 μl RD reaction + 1 μl dH2O + 1 μl 5xLD

- loading of 1 μl uncut plasmid + 3 μl dH2O + 1 μl 5xLD

- rut at 100 V

- EtBr staining + destaining in water

- UV detection

loading: Marker/DarRrev-Termrev C3 plasmid/ part 6.4 A C3 uncut plasmid (from today’s prep)/ part 6.4 A C3 vector (E + X)/part 6.4 B C2 uncut plasmid (from today’s prep)/ part 6.4 B C2 vector (E + X)/ part 6.4 B C5 plasmid/Marker

→ for vector digests, the expected bands were obtained (ca. 3 kb), but the digestes are still slightly incomplete (weak 2 kb band) → dephosphorylation with AP

→ plasmids purified today look normal

Dephosphorylation of part 6.4 A and part 6.4 B vector with AP

ca. 37 μl digest reaction

+ 2 μl AP

+ 5 μl 10x AP buffer

+ 6 μl dH2O

incubation for 1 h at 37 °C (EcoRI and XbaIform5’ overhang, but dephosphorylation often incomplete → incubation for more than 15 min)

Test RD of Promoter1rev and Promoter3rev in pSB1C3

2 μl plasmid (from today’s purification, ca. 200 – 300 ng plasmid)

1 μl EcoRI FD

1 μl PstI FD

1 μl 10x FD Green buffer

5 μl dH2O

in total: 10 μl

- both reactions were pipetted individually

- incubation for 1 h at 37 °C

Gel run: Test RD of Promoter1rev and Promoter3rev in pSB1C3

- 1 % agarose-1x TAE gel

- loading of 3 μl 2 log ladder

- loading of 5μl RD reaction

- loading of 1 μl uncut plasmid + 3 μl dH2O + 1 μl 5xLD

- rut at 100 V

- EtBr staining + destaining in water

- UV detection

Loading: marker/uncut Promoter1rev C4 plasmid/ RD Promoter1rev C4 plasmid/ uncut Promoter3rev C5 plasmid/ RD Promoter3rev C5 plasmid

→ both digests are only partial

→ but the expected bands were obtained: 112 bp Promrev + 2x20 bp prefix/suffix = 152 bp and ca. 2 kb of pSB1C3 backbone

→ sequencing of both plasmids

PCR clean-up of vectors (part 6.4 A and part 6.4 B vector; DarRrev-Termrev C3 vector) after AP treatment

- with Qiagen PCR purification kit

- addition of 500 μl PB buffer

- elution with 30 μl HPLC water, incubating for 2 min at 50 °C, centrifugation by mistake for 2 min at 13 000 rpm

- NanoDrop concentration measurement:

Plates Prepared for the Drop experiments

4 plates:

1x Gp1013 without IPTG

1x Gp1013 with IPTG

1x p172 without IPTG

1x p172 with IPTG

As follows:

25ml sterile water were filled intotwo 50ml falcons. Also two of the 300µl Glycerin stocks (Gp1013 and p172) prepared earlier were added to the water in the dedicated falcons. The falcons were inverted several times. 2x LB medium was prepared by Katrin Gunka and melted in the microwave. It was added to the 25ml water rather hot to the total volume of 50ml. The falcons were inverted a few times and then one 25ml plate was poured from each falcon. Then, very fast, where needed, 25µl IPTG 1M (end concentration 1mM) were added.

Inoculation of Promoter clones for Plate Reader Assay and RT-PCR analysis

- inoculation of

part 1 C1

part 2 C1

part 2 C2

part 3 C1

part 4 C1

part 8 C1 (as empty vector control, AmpR, but no RFP, only RBS)

- in LBAmp from cryostocks:(scratch some frozen E.coli cells from culture tube with a yellow pipet tip and transfer them directly in LBAmp)

- incubation ON at 37 °C at 200 – 210 rpm

Fold ↑

26th

Sequencing results from 23.8.13, Restriction digests, Purification of vectors from today by PCR clean-up after first round of digest, Purification of part 6.4 A/B inserts by gel extraction....

Sequencing results from 23.8.13

9 – DarRrev-Termrev C2 + VF2→ DarRrev and Terminatorrev are inserted in the plasmid in the desired orientation without mutations

10 – DarRrev-Termrev C2 + VR→ DarRrev and Terminatorrev are inserted in the plasmid in the desired orientation without mutations

11 – DarRrev-Termrev C3 + VF2→ DarRrev and Terminatorrev are inserted in the plasmid in the desired orientation without mutations

12 - DarRrev-Termrev C3 + VR→ DarRrev and Terminatorrev are inserted in the plasmid in the desired orientation without mutations

13 – part6.4 B C2 + VF2→ RBSrev is inserted in the desired orientation, but sequencing stopped again in reverse promoter 3

14 – part 6.4 B C5 + VF2→ RBSrev is inserted in the desired orientation, but sequencing stopped again in reverse promoter 3

15 – part 6.4 A C2 + VF2 → RBSrev is not inserted

16 – part 6.4 A C3 + VF2→ RBSrev is inserted in the desired orientation, but a part of pSB1C3 is missing at the EcoRI restriction site (but EcoRI restriction site is present) and a G of NotI restriction site in prefix is missing…

17 - part 6.4 A C5 + VF2→ RBSrev is not inserted

18 – part 6.4 A C8 + VF2→ RBSrev is not inserted

Plan: sequencing of the other 4 part 6.4 A clones and cutting out of part 6.4 A C3 insert with XbaI and PstI (one would get rid of missing/erroneous regions) and integration in vector DarRrev-Termrev plasmid C3; same for part 6.4 B C2

Plates for C1 – C3 of parts 1 – 4

- today, the part 2 clones C2 and C3 are slightly pink, while C1 is white as C1 and C2 of part 1 (C3 of part 1 never existed…)

- clones of part 3 and part 4 are very pink

- pictures were taken:

Restriction digests

a) DarRrev-Termrev in pSB1C3; C3 with EcoRI and SpeI

3 μl SpeI FD

3 μl EcoRI FD

4 μl FD buffer 10x

7 μl plasmid (229.7 ng/μl, purified on 23.8.13 → ca. 1500 ng)

23 μl dH2O

in total: 40 μl

b) part 6.4 A C3 and part 6.4 B C2 with EcoRI

4μl EcoRI FD

4 μl FD buffer 10x

15μl plasmid (6.4 A: 107.5 ng/μl, purified on 23.8.13 → ca. 1500 ng; 6.4 B: 114.6 ng/μl, purified on 23.8.13 → ca. 1500 ng)

17μl dH2O

in total: 40 μl

Cloning strategy changed - suddenly new idea occurred to Katrin and me: sequencing of the other 4 part 6.4 A clones and cutting out of part 6.4 A C3 insert with XbaI and PstI and integration in vector DarRrev-Termrev plasmid C3; same for part 6.4 B C2; but to avoid a waste of material, the above mentioned reactions were incubated as well and stored at – 20°C in DarR box (in addition, they can serve as a control for part 6.4A, to see, if EcoRI site is really there!)

c) DarRrev-Termrev in pSB1C3; C3 with SpeI

4μl SpeI FD

4 μl FD buffer 10x

7 μl plasmid (229.7 ng/μl, purified on 23.8.13 → ca. 1500 ng)

25μl dH2O

in total: 40 μl

d) part 6.4 A C3 and part 6.4 B C2 with XbaI and PstI

3μl PstI FD

3 μl XbaI FD

4 μl FD buffer 10x

15μl plasmid (6.4 A: 107.5 ng/μl, purified on 23.8.13 → ca. 1500 ng; 6.4 B: 114.6 ng/μl, purified on 23.8.13 → ca. 1500 ng)

15μl dH2O

in total: 40 μl

→ all reactions incubated for 1.5 h at 37 °C (incubation time decreased since missing part of vector in plasmid part 6.4 A C3 might result from EcoRI FD overdigest within 2 h incubation time…)

Test Gel run for Restriction digests (see above)

- 1 % agarose-1x TAE gel

- loading of 3 μl 2 log ladder

- loading of 3 μl RD reaction + 1 μl dH2O + 1 μl 5xLD

- loading of 1 μl uncut plasmid + 3 μl dH2O + 1 μl 5xLD

- rut at 100 V

- EtBr staining + destaining in water

- UV detection

Gel:

Loading: Marker/ part 6.4 A C3 uncut/ part 6.4 A C3 RD EcoRI/ part 6.4 A C3 RD XbaI + PstI/ part 6.4 B C2 uncut/ part 6.4 B C2 RD EcoRI/ part 6.4 B C2 RD XbaI + PstI/DarRrevTermrev C3 uncut/ DarRrevTermrev C3 RD EcoRI + SpeI/ DarRrevTermrev C3 RD SpeI/Marker

→ both digests of part 6.4 A seemed to be complete (expected bands for XbaI/PstI double digest were obtained, i.e. ca. 1 kb and ca. 2 kb band; expected band for EcoRI single digest, i. e. 3 kb band, was also obtained)

→ single digest of part 6.4 B seemed to be slightly incomplete, while double digest appeared to be complete (expected bands for XbaI/PstI double digest were obtained, i.e. ca. 1 kb and ca. 2 kb band; expected band for EcoRI single digest, i. e. 3 kb band, was also obtained)

→ single digest of DarRrev-Termrev seemed to be slightly incomplete, while double digest appeared to be complete (expected bands for EcoRI/SpeI double digest were obtained, i.e. ca. 0.8 kb and ca. 2 kb band; expected band for SpeI single digest, i. e. ca. 2.8 kb band, was also obtained)

- purification of inserts (except for DarRrev-Termrev insert, stored unpurified at – 20 °C in To-do-box) by gel ex and purification of vectors by PCR clean-up

Purification of vectors from today by PCR clean-up (DarRrev-Termrev vector/part 6.4 A/B vector) after first round of digest

- with Qiagen PCR purification kit

- 500 μl PB buffer were added to the samples

- elution with 30 μl pre-warmed HPLC water, incubation for 2 min at 50 °C

- further digest

Second round of restriction digest to generate vectors for ligation

a) DarRrev-Termrev vector previously cut with SpeI

30 μl linearized plasmid

2 μl dH2O

4 μl PstI FD

4 μl FD buffer 10x

in total: 40 μl

b) part 6.4 A/B vector previously cut with EcoRI

30 μl linearized plasmid

2 μl dH2O

4 μl XbaI FD

4 μl FD buffer 10x

in total: 40 μl

- incubation of all three reactions at 37 °C for 1.5 h

- samples stored at – 20 °C in to-do-box

Purification of part 6.4 A/B inserts by gel extraction

- 1 % agarose-1x TAE gel

- loading of 3 μl 2 log ladder

- loading of entire RD reaction (ca. 37 μl supplied with 7μl 5xLD)

- loading of 1 μl uncut plasmid + 3 μl dH2O + 1 μl 5xLD

- rut at 85 V

- brief EtBr staining + brief destaining in water

- short UV detection, then gel extraction as described on 25.6.13

gel after gel ex:

loading: Marker/ uncut part 6.4 A C3/ - /RD part 6.4 A C3/ RD part 6.4 A C3/ RD part 6.4 A C3/ RD part 6.4 A C3/ RD part 6.4 A C3/-/RD part 6.4 B C2/ RD part 6.4 B C2/ RD part 6.4 B C2/ RD part 6.4 B C2/uncut part 6.4 B C2/ Marker

Inoculation of clones for CryoStocks and MiniPrep for Test RD/Sequencing

inoculation of

- part 1 C2

- part 2 C3

- part 3 C2 and C3

- part 4 C2 and C3

→ in 4 ml LBAmp

→ all for cryo stocks and MiniPrep

inoculation of

- DarRrev-Termrev in pSB1C3 C3 → for cryostock and MiniPrep

- part 6.4 A C3 → for cryostock and MiniPrep

- part 6.4 B C2 and C5 → for cryostock and MiniPrep

- Promoter1rev in pSB1C3 C4 → for cryostock, MiniPrep, test RD and sequencing

- Promoter3rev in pSB1C3 C5 → for cryostock, MiniPrep, test RD and sequencing

→ in 4 ml LBCm

incubation ON at 37 °C, 200 - 210 rpmpurification: no isopronol used since fragments ca. 1 kb; each sample (part 6.4 A or part 6. 4 B) was distributed onto two colums, DNA from each column was eluted with 30 μl pre-warmed HPLC water (incubation of sample for 2 min at 50 °C. The DNA from both columns of one sample was collected in the same tube.

- NanoDrop concentration measurement:

sample

concentration (ng/μl)

A260nm/A280nm

A260nm/A230nm

part 6.4 A C3 insert

+ P

6.0

1.51

0.07

part 6.4 B C2 insert X + P

6.5

1.66

0.06

- sample stored in To-do-box

Inocculation of Ribo A C5.6 and C5.8 from cryo stocks in LB medium containing Cm

Inoccultion of DAC (GP1013) and the empty vector (pGP172) from cryo stocks in LB medium containing Amp

both stored at 30°C over night in the shaker

Fold ↑

@@ Line 54: / Line 54: @@
 .c1353{margin-left:48.8pt}
 </style>
-<div class="monat">August</div>
+<div class="monat">August</div><div class="tlob" id="tl_0827">
-<div class="tlob" id="tl_0827">
 <span class="date">27th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
 <div class="cont">
-<p class="timeline-title">Test gel for DarRrev-Termrev C3 vector after 2nd digest (with PstI) and dephosphorylation, Plasmid Mini Preparation, Test gel for plasmids after Plasmid Mini Preparation and part 6.4 A/B vectors....</p>
+<p class="timeline-title">Test gel for DarR<sub>rev</sub>-Term<sub>rev</sub> C3 vector after 2nd digest (with PstI) and dephosphorylation, Plasmid Mini Preparation, Test gel for plasmids after Plasmid Mini Preparation and part 6.4 A/B vectors....</p>
 <div class="timeline-cont">
 <p class="c133"><span class="c135">Test gel for DarR</span><span class="c135 c131">rev</span><span class="c135">-Term</span><span class="c135 c131">rev</span><span class="c135">&nbsp;C3 vector after 2</span><span class="c135 c1331">nd</span><span class="c135">&nbsp;digest (with PstI) </span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>1 % agarose-1x TAE gel</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>loading of 3 μl 2 log ladder</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>loading of 3 μl RD reaction + 1 μl dH</span><span class="c131">2</span><span>O + 1 μl 5xLD</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>loading of 1 μl uncut plasmid + 3 μl dH</span><span class="c131">2</span><span>O + 1 μl 5xLD</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>rut at 100 V</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>EtBr staining + destaining in water</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>UV detection</span><span class="c135">&nbsp;</span></p><p class="c133"><span class="c135">loading: </span><span>Marker/uncut DarR</span><span class="c131">rev</span><span>-Term</span><span class="c131">rev</span><span>&nbsp;C3 plasmid/ RD DarR</span><span class="c131">rev</span><span>-Term</span><span class="c131">rev</span><span>&nbsp;C3 vector</span></p><img src="https://static.igem.org/mediawiki/2013/7/7e/Goe-27.08.13-RT-1.png" /><p class="c133"><span>digest still partial (expected band at ca. 3kb is observed, but also a weak band at ca. 6 – 8 kb of uncut plasmid) → dephosphorylation of vector with AP to avoid self-ligation</span></p><p class="c1318 c133"><span></span></p><p class="c133"><span class="c135">Preparation of LB media </span></p><p class="c133 c1328"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>1x 300 ml broth LB w/o antibiotics</span></p><p class="c133 c1328"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>1x 500 ml LB</span><span class="c1331">Cm</span><span>&nbsp;plates</span></p><p class="c1318 c133"><span class="c135"></span></p><p class="c133"><span class="c135">Dephosphorylation of DarR</span><span class="c135 c131">rev</span><span class="c135">-Term</span><span class="c135 c131">rev</span><span class="c135">&nbsp;C3 vector cut with SpeI and PstI </span></p><p class="c133"><span>ca. 37 μl digest reaction</span></p><p class="c133"><span>+ 2 μl AP</span></p><p class="c133"><span>+ 5 μl 10x AP buffer</span></p><p class="c133"><span>+ 6 μl dH</span><span class="c131">2</span><span>O </span></p><p class="c133"><span>incubation for 1.5 h at 37 °C (PstI forms 3’ overhang, but dephosphorylation often incomplete → incubation for more than 1 h)</span></p><p class="c1318 c133"><span class="c135"></span></p><p class="c133"><span class="c135">Preparation of CryoStocks of clones inoculated yesterday </span></p><p class="c133"><span>DMSO cryostocks prepared as described before (13.08.2013)</span></p><p class="c133"><span>for all clones inoculated on 26.8.13:</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>part 1 C2</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>part 2 C3</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>part 3 C2 and C3</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>part 4 C2 and C3 </span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>DarR</span><span class="c131">rev</span><span>-Term</span><span class="c131">rev</span><span>&nbsp;in pSB1C3 C3</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>part 6.4 A C3</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>part 6.4 B C2 and C5</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>Promoter1</span><span class="c131">rev</span><span>&nbsp;in pSB1C3 C4</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>Promoter3</span><span class="c131">rev</span><span>&nbsp;in pSB1C3 C5</span></p><p class="c1318 c133"><span class="c135"></span></p><p class="c133"><span class="c135">Plasmid Mini Preparation</span><span>&nbsp;</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>harvesting of all cultures inoculated yesterday </span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>but MiniPrep only for the following clones:</span></p><p class="c133 c1310"><span>DarR</span><span class="c131">rev</span><span>-Term</span><span class="c131">rev</span><span>&nbsp;in pSB1C3 C3</span></p><p class="c133 c1310"><span>part 6.4 A C3</span></p><p class="c133 c1310"><span>part 6.4 B C2 and C5</span></p><p class="c133 c1310"><span>Promoter1</span><span class="c131">rev</span><span>&nbsp;in pSB1C3 C4</span></p><p class="c133 c1310"><span>Promoter3</span><span class="c131">rev</span><span>&nbsp;in pSB1C3 C5</span></p><p class="c133 c1310"><span>&nbsp;</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>all other clones were harvested and the cell pellets stored in to-do-box (it could be that we won’t need the plasmids…):</span></p><p class="c133 c1310"><span>part 1 C2</span></p><p class="c133 c1310"><span>part 2 C3</span></p><p class="c133 c1310"><span>part 3 C2 and C3</span></p><p class="c133 c1310"><span>part 4 C2 and C3 </span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>elution 1x with 30 μl HPLC water (pre-warmed), incubation at 50 °C for 2 min</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>NanoDrop concentration measurement:</span></p><img src="https://static.igem.org/mediawiki/2013/8/8e/Goe-27.08.13-RT-2.png" /><p class="c138 c1318 c133"><span></span></p><p class="c138 c133"><span class="c135">Test gel for plasmids after Plasmid Mini Preparation and part 6.4 A/B vectors </span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>1 % agarose-1x TAE gel</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>loading of 3 μl 2 log ladder</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>loading of 3 μl RD reaction + 1 μl dH</span><span class="c131">2</span><span>O + 1 μl 5xLD</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>loading of 1 μl uncut plasmid + 3 μl dH</span><span class="c131">2</span><span>O + 1 μl 5xLD</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>rut at 100 V</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>EtBr staining + destaining in water</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>UV detection</span><span class="c135">&nbsp;</span></p><p class="c138 c133"><span class="c135">loading: </span><span>Marker/DarR</span><span class="c131">rev</span><span>-Term</span><span class="c131">rev</span><span>&nbsp;C3 plasmid/ part 6.4 A C3 uncut plasmid (from today’s prep)/ part 6.4 A C3 vector (E + X)/part 6.4 B C2 uncut plasmid (from today’s prep)/ part 6.4 B C2 vector (E + X)/ part 6.4 B C5 plasmid/Marker</span></p><img src="https://static.igem.org/mediawiki/2013/b/b5/Goe-27.08.13-RT-3.png" /><p class="c1325 c133 c1328"><span>→ </span><span>for vector digests, the expected bands were obtained (ca. 3 kb), but the digestes are still slightly incomplete (weak 2 kb band) → dephosphorylation with AP</span></p><p class="c1325 c133 c1328"><span>→ </span><span>plasmids purified today look normal</span></p><p class="c1318 c133"><span class="c1315"></span></p><p class="c133"><span class="c135">Dephosphorylation of part 6.4 A and part 6.4 B vector with AP </span></p><p class="c133"><span>ca. 37 μl digest reaction</span></p><p class="c133"><span>+ 2 μl AP</span></p><p class="c133"><span>+ 5 μl 10x AP buffer</span></p><p class="c133"><span>+ 6 μl dH</span><span class="c131">2</span><span>O </span></p><p class="c133"><span>incubation for 1 h at 37 °C (EcoRI and XbaIform5’ overhang, but dephosphorylation often incomplete → incubation for more than 15 min)</span></p><p class="c1318 c133"><span class="c135"></span></p><p class="c133"><span class="c135">Test RD of Promoter1</span><span class="c135 c131">rev</span><span class="c135">&nbsp;and Promoter3</span><span class="c135 c131">rev</span><span class="c135">&nbsp;in pSB1C3</span><span>&nbsp;</span></p><p class="c133"><span>2 μl plasmid (from today’s purification, ca. 200 – 300 ng plasmid)</span></p><p class="c133"><span>1 μl EcoRI FD</span></p><p class="c133"><span>1 μl PstI FD</span></p><p class="c133"><span>1 μl 10x FD Green buffer</span></p><p class="c133"><span>5 μl dH</span><span class="c131">2</span><span>O</span></p><p class="c133 c1317 c1310"><span>in total: 10 μl </span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>both reactions were pipetted individually</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>incubation for 1 h at 37 °C</span></p><p class="c1318 c133"><span class="c135"></span></p><p class="c133"><span class="c135">Gel run: Test RD of Promoter1</span><span class="c135 c131">rev</span><span class="c135">&nbsp;and Promoter3</span><span class="c135 c131">rev</span><span class="c135">&nbsp;in pSB1C3 </span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>1 % agarose-1x TAE gel</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>loading of 3 μl 2 log ladder</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>loading of 5μl RD reaction</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>loading of 1 μl uncut plasmid + 3 μl dH</span><span class="c131">2</span><span>O + 1 μl 5xLD</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>rut at 100 V</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>EtBr staining + destaining in water</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>UV detection </span></p><p class="c133"><span class="c135">Loading</span><span>: marker/uncut Promoter1</span><span class="c131">rev</span><span>&nbsp;C4 plasmid/ RD Promoter1</span><span class="c131">rev</span><span>&nbsp;C4 plasmid/ uncut Promoter3</span><span class="c131">rev</span><span>&nbsp;C5 plasmid/ RD Promoter3</span><span class="c131">rev</span><span>&nbsp;C5 plasmid</span></p><img src="https://static.igem.org/mediawiki/2013/e/e5/Goe-27.08.13-RT-4.png" /><p class="c1325 c133 c1328"><span>→ </span><span>both digests are only partial</span></p><p class="c1325 c133 c1328"><span>→ </span><span>but the expected bands were obtained: 112 bp Prom</span><span class="c131">rev</span><span>&nbsp;+ 2x20 bp prefix/suffix = 152 bp and ca. 2 kb of pSB1C3 backbone</span></p><p class="c1325 c133 c1328"><span>→ </span><span>sequencing of both plasmids</span></p><p class="c1318 c133"><span></span></p><p class="c133"><span class="c135">PCR clean-up of vectors (part 6.4 A and part 6.4 B vector; DarR</span><span class="c135 c131">rev</span><span class="c135">-Term</span><span class="c135 c131">rev</span><span class="c135">&nbsp;C3 vector) after AP treatment </span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>with Qiagen PCR purification kit</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>addition of 500 μl PB buffer</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>elution with 30 μl HPLC water, incubating for 2 min at 50 °C, centrifugation by mistake for 2 min at 13 000 rpm</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>NanoDrop concentration measurement:</span></p><img src="https://static.igem.org/mediawiki/2013/7/7c/Goe-27.08.13-RT-5.png" /><p class="c1318 c133"><span></span></p><p class="c133"><span class="c135">Plates Prepared for the Drop experiments </span></p><p class="c133"><span class="c135">4 plates: </span></p><p class="c133"><span>1x Gp1013 without IPTG</span></p><p class="c133"><span>1x Gp1013 with IPTG</span></p><p class="c133"><span>1x p172 without IPTG</span></p><p class="c133"><span>1x p172 with IPTG</span><span class="c135">&nbsp;</span></p><p class="c133"><span>As follows: </span></p><p class="c133"><span>25ml sterile water were filled intotwo 50ml falcons. Also two of the 300µl Glycerin stocks (Gp1013 and p172) prepared earlier were added to the water in the dedicated falcons. The falcons were inverted several times. 2x LB medium was prepared by Katrin Gunka and melted in the microwave. It was added to the 25ml water rather hot to the total volume of 50ml. The falcons were inverted a few times and then one 25ml plate was poured from each falcon. Then, very fast, where needed, 25µl IPTG 1M (end concentration 1mM) were added.</span></p><p class="c1318 c133"><span class="c135"></span></p><p class="c133"><span class="c135">Inoculation of Promoter clones for Plate Reader Assay and RT-PCR analysis </span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>inoculation of</span></p><p class="c133 c1310"><span>part 1 C1</span></p><p class="c133 c1310"><span>part 2 C1</span></p><p class="c133 c1310"><span>part 2 C2</span></p><p class="c133 c1310"><span>part 3 C1</span></p><p class="c133 c1310"><span>part 4 C1</span></p><p class="c133 c1310"><span>part 8 C1 (as empty vector control, AmpR, but no RFP, only RBS)</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>in LB</span><span class="c1331">Amp</span><span>&nbsp;from cryostocks:(scratch some frozen E.coli cells from culture tube with a yellow pipet tip and transfer them directly in LB</span><span class="c1331">Amp</span><span>)</span></p><p class="c133 c1317 c1310"><span>-</span><span>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; </span><span>incubation ON at 37 °C at 200 – 210 rpm</span></p><p class="c1318 c133"><span></span></p>
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