Team:Goettingen/NoteBook w12

Cloning of Riboswitch A

Mini-Preps of RiboA Clones 1, 2, 4 and 5 (see gel from 12.08.) using the plasmid purification kit

Nanodrop measurements:

Restriction Digestion of

Ribo A clones 4 +5, (R.D. with E + S)

Ribo B clone 2, (R.D. with X + P)

Ribo C clone 4, (R.D. with E + S)

Ribo D clone 5 (R.D. with X + P)

1500 ng DNA

3µl enzyme FD

3µl enzyme FD

4µl Buffer FD

40 µl reaction (filled up with H2O)

for 1.5 h at 37°C

Gel:

M/plasmid DNA Ribo A clone 4/ R.D. Ribo A clone 4/ plasmid DNA Ribo A clone 5/ R.D. Ribo A clone 5/ plasmid DNA Ribo B clone 2/ R.D. Ribo B clone 2/ plasmid DNA Ribo C clone 4/ R.D. Ribo C clone 4/ plasmid DNA Ribo D clone 5/ R.D. Ribo D clone 5

→ loading of all R.D. Ribos A-D onto Gel for gel extraction

→ Gelextraxtion using the PCR purification kit, nanodrop measurements on the eppis (3-5ng/µl)

Ligations of the Riboswitches into next vectors

Ribo A clones 4 +5, (R.D. with E + S) with CFP and YFP (R.D. with E+X) (see 12.08.)

Ribo B clone 2, (R.D. with X + P) with Part1 and Part3 (R.D. with S+P) (see 12.08.)

Ribo C clone 4, (R.D. with E + S) with Part8 (R.D. with E+X) (see 12.08.)

Ribo D clone 5 (R.D. with X + P) with Part1 and Part3 (R.D. with S+P) (see 12.08.)

For that, all of the inserts (60ng) was used for ligation ino the Vectors (20ng)

0,75-1µl Vectors

12µl Insert (Ribo A-D)

2µl T4 Buffer

2µl T4 Ligase

3-3,25µl H20

20µl reaction

Incubation overnight at 16°C

MiniPrep of RBSrev + pSB1C3, Prom1rev + part6.2, Prom3rev + part 6.2 (C1 – C3 each)

-          kit: Nucleospin, Macherey-Nagel

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

-          2nd elution: 22 μl HPLC-water (pre-warmed), incubation for 2 min at 50 °C

-          no concentration measurement, since NanoDrop is dirty and measures 6 – 7 ng DNA in HPLC water used as a blank before…

-          samples stored in “DarR reporter system” - box

PCR to test, if RBSrev C1 – C3 are positive or negative 

-          RBSrev insert has only 12 bp →  one would not see it in a gel

-          in Colony PCR, there was for all clones a band at height of terminator band (re-ligand)

-          perform PCR to test, if terminator band is really there/ if clones are positive

-          PCR (similar to colony PCR, but with plasmids as a template):

-          of the plasmids prepped today (see MiniPrep), a 1:20 dilution was prepared, of this dilution, 1 μl was used a template (1 reaction for each clone)

-          as an additional control, part 7 C1 plasmid (dilution from 15.6.13, 1:10) was used

-          Neg. control: 1 μl water as a template

-          preparation of a 6x MasterMix:

→ addition of 24 μl MasterMix to each sample

-          PCR protocol: same as for colony-PCR

Test restriction digest of Prom1rev + part6.2, Prom3rev + part 6.2 (C1 – C3 each) 

-          of the plasmids prepped today, 2 μl were used for the digest, since the concentration could not be measured with NanoDrop

-          one reaction contained

-          the plasmid was added to the tubes, then addition of 8 μl MasterMix; the MasterMix consisted of…

-          incubation at 37 °C for 1 h

Gel run:

-          1 % agarose-1xTAE gel

-          loading of 3 μl 2 log ladder

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

-          loading of 5 μl of RD reaction

-          run at 100 V

-          EtBr staining + destaining

-          UV detection

 Loading: Marker/Prom1rev + part6.2 C1 uncut/ Prom1rev + part6.2 C1 RD/ Prom1rev + part6.2 C2 uncut/ Prom1rev + part6.2 C2 RD/ Prom1rev + part6.2 C3 uncut/ Prom1rev + part6.2 C3 RD/ Prom3rev + part6.2 C1 uncut/ Prom3rev + part6.2 C1 RD/ Prom3rev + part6.2 C2 uncut/ Prom3rev + part6.2 C2 RD/ Prom3rev + part6.2 C3 uncut/ Prom3rev + part6.2 C3 RD/Marker

part 6.2 Re-ligand: 35 bp (Promoter 3) + 8 bp (Scar between promoter and DarR operator) + 14 bp (operator) + 8 bp (Scar between operator and part 6 GFP) + 876 bp (part 6 GFP) = 941 → ca. 940 bp should be visible in addition to pSB1C3 (2070 bp)

part 6.2 C1 plasmid and Promoter1rev/Promoter3rev: 112 bp (rev. Promoter) + 8 bp (Scar between rev. promoter and promoter 3) + 35 bp (Promoter 3) + 8 bp (Scar between promoter and DarR operator) + 14 bp (operator) + 8 bp (Scar between operator and part 6 GFP) + 876 bp (part 6 GFP) = 1061 → ca. 1060 bp should be visible in addition to pSB1C3 (2070 bp)

Conclusion:

All clones seemed to be positive, since fragments have slightly more than 1 kb and ca. 2 kb. Sequencing of C1 and C2 of each.

Sequencing results from 16.8.13

Terminatorrev + pSB1C3 C1 and C3: both clones contain the correct sequence of the terminator, but in reverse direction

DarRrev + pSB1C3 C1:

-          between NotI and PstI restriction site, an additional G is inserted

-          2nd base of 10th codon: A is missing

-          → don’t work with this clone! 

DarRrev + pSB1C3 C4:

-          all mutations from the forward primer are present at correct site

-          no other mutations observed

-          →  work with this clone

Restriction of Terminatorrev + pSB1C3 C1 for generating vector for ligation with DarRrev 

15 μl (ca. 1500 ng) plasmid Termrev C1 (from 16.8.13, 102.8 ng/μl)

4 μl SpeI FD

4 μl FD buffer 10x

17 μl dH2O

in total 40 μl

-         digest: 2 h at 37 °C

Restriction of DarRrev + pSB1C3 C4 for generating the insert for ligation with Termrev C1 vector

6 μl (ca. 1500 ng) plasmid Termrev C1 (from 16.8.13, 282.5 ng/μl)

3μl XbaI FD

3 μl PstI FD

4 μl FD buffer 10x

24μl dH2O

in total 40 μl

-         digest: 2 h at 37 °C

Gel run for DarRrev + pSB1C3 C4 and Terminatorrev + pSB1C3 C1 RDs and RBSrev Test PCRs (all from today, see above) 

-          1 % agarose-1xTAE gel

-          loading of 3 μl 2 log ladder

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

-          loading of 5 μl of RD reaction

-          loading of 5 μl PCR reaction (addition of 5 μl of 5xLD to each PCR sample)

-          run at 100 V

-          EtBr staining + destaining

-          UV detection

Loading: Marker/Terminatorrev C1 uncut/Terminatorrev + SpeI RD/DarRrev C4 uncut/DarRrev C4 + XbaI and PstI/RBSrev PCR: neg. control/RBSrev PCR: part 7 C1 plasmid/RBSrev PCR: RBSrev C1/RBSrev PCR: RBSrev C2/RBSrev PCR: RBSrev C3/Marker

-          Termrev C1 is digested incompletely, but since the DarRrev insert won’t be dephosphorylated, one can dephosphorylate the incompletely digested vector →  purifiy vector and digest with PstI

-          DarRrev C4 plasmid is digested completely →  purify DarRrev fragment with gel extraction

-          PCR: the plasmids part 7 C1 and the RBSrev C1, C2 and C3 showed the expected products, but the negative control had a band at ca. 200 – 300 bp (I’ve no explanation for that: the water I used was the same for all other reactions! And the other material as well. Could be Bacillus spores, unclean PCR tubes, or my DNA…? XD →  forget negative control (some people even don’t do no-template controls…), since expected products dominate/bands on neg. control seem not to be in lanes of all other samples →  prepare C1 for sequencing

Hybridization of RBSrev, Promoter1rev and Promoter3rev repeated  

-          RBSrev must be  cloned into Prom1rev/Prom3rev + part6.2 (from now on termed part 6.3A/B)

-          Promoter1rev and Promoter3rev must be cloned into shipping vector

-          hybridization performed as on 13.08.2013

-          samples stored at – 20 °C in DarR reporter system-box

Concentration measurement (NanoDrop worked again!) of plasmids purified today

Preparation of samples for sequencing by SeqLab

-          sample no. 3: Promoter1rev + part6.2 C1 (part 6.3 A C1) + VF2 (6 μl plasmid, purified today + 1 μl primer iGEM_38 1:5)

-          sample no. 4: Promoter1rev + part6.2 C2 (part 6.3 A C1) + VF2 (6 μl plasmid, purified today + 1 μl primer iGEM_38 1:5)

-          sample no. 5: Promoter3rev + part6.2 C1 (part 6.3 B C1) + VF2 (6 μl plasmid, purified today + 1 μl primer iGEM_38 1:5)

-          sample no. 6: Promoter3rev + part6.2 C2 (part 6.3 B C1) + VF2 (6 μl plasmid, purified today + 1 μl primer iGEM_38 1:5)

-          sample no. 7: RBSrev + pSB1C3 C1 + VF2 (6 μl plasmid, purified today + 1 μl primer iGEM_38 1:5)

PCR clean up of Termrev C1 + SpeI digest

-          with Qiagen PCR purification kit

-          500 μl PB buffer used

-          elution 1x with 30 μl HPLC water (pre-warmed), incubation at ca. 57°C for 2 min

sample stored at – 20 °C in DarR reporter system box, marked with green

Gel ex of DarRrev insert 

-          1%-agarose-1xTAE gel

-          addition of 9 μl 5x LD to 37 μl reaction

-          loading of whole reaction on gel

-          loading of 3 μl marker

-          loading of 1 μl uncut DarRrev C4 plasmid + 3 μl dH2O + 1 μl 5xLD

-          run at 85 V

-          5 min staining with EtBr, short destaining in water, UV detection briefly, then gel ex under low UV light, tried to do gel ex, but signal was too weak, so again brief staining and destaining, then gel ex was possible

Gel after gel ex:

-          a bit was lost, but that’s ok…

-          gel pieces weighed and stored in DarR reporter system box at – 20 °C