Team:Goettingen/NoteBook w13

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 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