Team:Goettingen/NoteBook w12

Transformation from 21.8.13

-          neg. control: no clones on 50 μl-plate, no clones on rest-plate

-          Termrev vector w/o insert: no clones on 50 μl-plate, few clones on rest-plate

-          Termrev vector + DarRrev insert: few clones on 50 μl-plate, many clones on rest-plate

-          part 6.3 A vector w/o insert: no clones on 50 μl-plate, 1 clone on rest-plate

-          part 6.3 A vector + RBSrev insert: no clones on 50 μl-plate, few clones on rest-plate

-          part 6.3 B vector w/o insert: no clones on 50 μl-plate, no clones on rest-plate

-          part 6.3 B vector + RBSrev insert: no clones on 50 μl-plate, some clones on rest-plate

-          pSB1C3 vector w/o insert: 1 or 2 clones on 50 μl-plate, several clones on rest-plate

-          pSB1C3 vector + Promoter1rev insert: no clones on 50 μl-plate, no clones on rest-plate

-          pSB1C3 vector + Promoter3rev insert: no clones on 50 μl-plate, few clones on rest-plate

→ colony PCR for DarRrev-Termrev transformation

→ further incubation of all other plates, until clones are bigger/grown

Colony PCR for DarRrev-Termrev transformation 

-          as described previously (10.7.13)

-          1x Termrev C1 plasmid as additional control (plasmid prepped on 16.8.13, 102.8 ng/μl →  1:10 dilution →  ca. 10 ng/μl), blue tube (termed T)

-          1x Re-ligand (termed R), pink tube

-          12x DarRrev-Termrev clones (termed C1 – C12), purple tubes

-          preparation of mastermix for 15 reactions, primers: iGEM_38 (VF2) and iGEM_39 (VR)

-          preparation of master plate (LBCm) →  incubation over day at 37 °C

-          25 μl mastermix + 1 μl plasmid dilution or cells

-          PCR protocol: same as on  10.7.13

-          plates stored at 4 °C, big fridge

Plates for part 1 – 4 C1 – C3 

-          plates taken out, since all clones grew

-          plates put to 4 °C, big fridge

Colony PCR for part6.3 B + RBSrev transformation 

-          after some additional incubation time

-          as described previously (10.7.13)

-          1x part6.3 B C2 plasmid as additional control (plasmid prepped on 19.8.13, 220.3 ng/μl →  1:20 dilution →  ca. 11 ng/μl), purple tube, termed 6.3 B

-          neg. control plates still without colonies

-          still no religands →  performing dephosphorylation longer than actually necessary seems to be helpful (see 20.8.13)

-          1 or 2 clones on 50 μl plate, many clones on rest-plate: 13x clones picked (termed C1 – C13), green tubes

-          preparation of mastermix for 15 reactions, primers: iGEM_38 (VF2) and iGEM_39 (VR)

-          preparation of master plate (LBCm) →  incubation over day at 37 °C

-          25 μl mastermix + 1 μl plasmid dilution or cells

-          PCR protocol: same as on  10.7.13

-          plates stored at 4 °C, big fridge

→ 12 bp (size of RBSrev) difference between positive and negative clones, one would never see in a gel… PCR thrown away…

Gel run for Colony PCR for DarRrev-Termrev transformation

-          1%-agarose-1xTAE gel

-          addition of 5 μl 5xLD to 25 μl PCR reaction

-          loading of 3 μl 2 log ladder as a marker

-          loading of 5 μl PCR reaction supplied with 5xLD

-          run at 100 V

-          EtBr staining + destaining in water

-          UV detection

Loading: Marker/Termrev plasmid/Re-ligand/ DarRrev-Termrev C1/ DarRrev-Termrev C2/ DarRrev-Termrev C3/ DarRrev-Termrev C4/ DarRrev-Termrev C5/ DarRrev-Termrev C6/ DarRrev-Termrev C7/ DarRrev-Termrev C8/ DarRrev-Termrev C9/ DarRrev-Termrev C10/ DarRrev-Termrev C11/ DarRrev-Termrev C12/Marker

-          expected product sizes

negative clone: 310 bp (VR, VF2) + 130 bp (Terminatorrev) = 440 bp

positive clone: 310 bp (VR, VF2) + 130 bp (Terminatorrev) + 630 bp (DarRrev) = 1070 bp

→ Religand plasmid corresponds to Termrev plasmid, as expected

→ all DarRrev-Termrev clones appear to be positive →  inoculate 3 clones for MiniPrep tomorrow

-          PCR reactions stored at 4 °C, small fridge

Sequencing of part6.3 B C1, C2 and C3 plasmids by G2L

sequencing by SeqLab failed; for both clones, sequence stopped at same region: possible reason: secondary structures formed during sequencing

5 μl total reaction: ca. 300 ng plasmid + 10 % DMSO (to prevent secondary structures) + 1 μl primer iGEM_38, 1:20 diluted

Preparation of the following samples

-          ppra_1: 2 μl plasmid prepped on 19.8.13 (268.4 ng/μl) + 1 μl DMSO (50 % in HPLC water) + 1 μl iGEM_38 1:20 + 1 μl HPLC water

-          ppra_2: 2 μl plasmid prepped on 19.8.13 (220.3 ng/μl) + 1 μl DMSO (50 % in HPLC water) + 1 μl iGEM_38 1:20 + 1 μl HPLC water

-          ppra_3: 2 μl plasmid prepped on 19.8.13 (247.2 ng/μl) + 1 μl DMSO (50 % in HPLC water) + 1 μl iGEM_38 1:20 + 1 μl HPLC water

-          ppra_4: 1 μl plasmid prepped on 21.8.13 (305.4 ng/μl) + 1 μl DMSO (50 % in HPLC water) + 1 μl iGEM_38 1:20 + 2 μl HPLC water

-          ppra_5: 1 μl plasmid prepped on 21.8.13 (332.1 ng/μl) + 1 μl DMSO (50 % in HPLC water) + 1 μl iGEM_38 1:20 + 2 μl HPLC water

-          ppra_6: 1 μl plasmid prepped on 21.8.13 (328.8 ng/μl) + 1 μl DMSO (50 % in HPLC water) + 1 μl iGEM_38 1:20 + 2 μl HPLC water

prior to sequencing, the samples have to be denatured →  remark was entered into excel-sheet

Plates of part6.3 A + RBSrev transformation

-          still no clones on neg. control

-          3 clones on w/o insert plate

-          8 clones on w/ insert plate

Inoculation of DarRrev-Termrev clones, part6.3 B + RBSrev(= part 6.4 B) and part 6.3 A + RBSrev(= part 6.4 A) clones for MiniPrep

-          inoculation of

DarRrev-Termrev clones C1 – C5→  Test restriction digest

 B clones C1 – C5 (no re-ligands)→  sequencing

part6.4 A clones C1 – C8 (3 re-ligands and 8 clones on WITH-insert plate→  sequencing

-          in 4 ml LBCm

-          incubation at 37 °C ON

Run the colony PCR from yesterday on a gel

Gel doc:

Wells: M/Ribo A C4 clone 1/2/3/4/5/6/7/8/9/10/blank/religant from control plate w/o insert

Wells: M/Ribo A C5 clone 1/2/3/4/5/6/7/8/9/10/blank/religant from control plate w/o insert

→ all clones with the high band should contain the Ribo A insert

Following clones were choosen to prepare a miniprep:

Ribo A C4 clones 3/4/6/7/8 (all others didn’t grew or showed wrong bands)

Ribo A C5 clones 2/3/6/8 (all others didn’t grew or showed wrong bands)

Miniprep of the DAC team Lm cryostock colony

Nanodrop:

Stored in to do box

Following plasmids were choosen for sequencing:

Again inocculation of all preped Ribo A C4 and C5 clones that were already prepped, for preparation of Cryostocks and a new miniprep, as the old one showed smears

Katrin Gunka

Preparation of plates with Lb medium also containing the DAC team Lm stain. One plate just like this, another one also containing

CryoStocks 

-          preparation of DMSO cryostocks of all inoculated clones (according to protocol described previously)

Plasmid Mini Prep

-          MiniPrep was done using the culture volume that remained after preparing the cryostocks (see above)

-          kit: Nucleospin, Macherey-Nagel

-          cells were harvested by centrifuging at 13000 g, 1 min

-          elution: 1x 30 μl HPLC water (pre-warmed), incubation for 3 min at 50 °C

-          NanoDrop concentration measurement:

Transformation of ligations prepared yesterday

-          according to protocol in methods folder

-          with XL1-Blue comp. cells

-          neg. control was done

-          when adding the Promoter1rev + pSB1C3 ligation mix to the comp. cells, a drop of the comp. cells was spilled…

-          500 μl were removed after centrifuging

-          plating on LBCm plates

-          incubation at 37 °C

Gel run after MiniPrep to check if plasmids are clean

-          1 %-agarose-1xTAE gel from yesterday (prepared by Jan)

-          loading of 3 μl 2 log ladder

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

-          run at 100 V

-          EtBr staining + destaining in water

-          UV detection

loading: Marker/Termrev C1/DarRrev C4/ RBSrev C1/part6.3 A C1/part6.3 B C1/ part6.3 B C2/ part6.3 B C3

→ all plasmids are purified from chrom.DNA and look normal

→ samples stored in DarR reporter system boxRe-streak of part 1 – 4 Clones 1 – 3 on LBAmp 

-          plates from 6.6.13 (stored in fridge…. at 4 °C….)

-          bacteria were quite dry, so I scratched some cell material off and streaked it out on new plates

-          incubation ON at 37 °C

-          let’s hope they grow…

Preparation of new LBCm plates 

-          2x500 ml

-          35 μg/ml Cm

-          stored at 4 °C, big fridge

observation of clones of Ribo A C4 and C5 CFP under UV light

(all clones with YFP don’t glow under UV light) 

Pictures:

Ribo A C4 + CFP UV light

Ribo A C4 + CFP transmitted light

Ribo A C5 + CFP UV light

Ribo A C5 + CFP transmiited light

Control w/o insert UV light

Control w/o insert transmitted light

Preparation of a Masterplate of 10 clones from plate with C4 and C5 CFP

Inocculation of 10 clones from plate with C4 and C5 CFP in LB medium with Cm

Colony PCR of the inoculated clones and a control w/o insert CFP

Transformation of yesterday´s ligation

Gel extraction of DarRrev insert 

-          with Qiagen PCR gel/PCR purification kit

-          since DarRrev insert has > 600 bp, no isopropanol was added/only QG buffer used

-          only 1 column used since <400 mg gel

-          elution 1x with 30 μl of pre-heated HPLC water, incubation at 50 °C for 2 min

-          NanoDrop concentration measurement

Restriction digest of Termrev with PstI FD

-          DNA: Termrev C1 plasmid restricted with SpeI and purified yesterday

-          digest: 

-          digest for 2 h at 37 °C

Restriction digest of RBSrev C1 and part6.3 A and B (C2 each) with EcoRI FD

-          DNA: plasmids purified yesterday

-          digest for all reactions (pipette individually, no mastermix):

 -          digest for 2 h at 37 °C

Sequencing

 results RBSrev C1 and part6.3 A and B (C1 and C2 each)

-          part 6.3 A C1 (sample 3) – sequence as expected (no mutations)

-          part 6.3 A C2 (sample 4) – a G at position 52 of Hybridization oligo is missing →  digest sample (see above) thrown away, new digest (exactly as described above) using clone 1

-          part 6.3 B C1 (sample 5) – sequencing did not work, only first part of oligo in vector sequenced →  was ok

-          part 6.3 B C2 (sample 6) – sequencing did not work, only first part of oligo in vector sequenced →  was ok →  further digest

-          RBSrev in pSB1C3 C1 (sample 7) – sequence as expected

Loading: 

Marker/uncut Termrev C1/RD Termrev C1/uncut RBSrev C1/RD RBSrev C1/uncut part6.3 A C1/RD part6.3 A C1/uncut part6.3 B C2/RD part6.3 B C2/Marker

-          all digests are only partial, but most of plasmid is cut  →  purify digests

New sizes of Riboswitch PCR products

I checked again the alignments of the riboswitch PCR products, and the sizes I entered into the journal on 24.6.13 are partially wrong.

The correct sizes are: 

Inoculation of clones for CryoStocks

inoculation of

-          RBSrev + pSB1C3 C1

-          part 6.3 A C1

-          part 6.3 B C1, C2, C3

-          Termrev + pSB1C3 C1

-          DarRrev + pSB1C3 C4

 → from master plates, inoculation in 4 ml LBCm

incubation ON at 37 °C, 200 – 210 rpm

Purification of vectors digested today 

-          samples: RBSrev + pSB1C3 RD, part 6.3 A C1 and part 6.3 B C2 RD, Termrev + pSB1C3 RD

-          with Qiagen PCR clean-up kit

-          addition of 500 μl PB buffer to each reaction

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

AP treatment of Termrev vector for ligation with DarRrev insert 

-          this is silly… I could have done it before purification, then I would have lost less vector…

-          addition of 1 μl AP, 4 μl AP buffer 10x, 8 μl dH2O to 27 μl purified vector

-          incubation for 1 h at 37 °C (PstI makes 3’ overhang)

Restriction Digest of all vectors digested with EcoRI FD today

-          samples: RBSrev + pSB1C3 RD, part 6.3 A C1 and part 6.3 B C2 RD

-          all samples had a volume of 30 μl after clean-up, so addition of 2 μl dH2O and 4 μl 10x FD buffer

-          addition of 4 μl SpeI FD to RBSrev + pSB1C3 RD sample, and 4μl XbaI FD to part 6.3 A C1 and part 6.3 B C2 RD samples

-          for all reactions: total volume of 40 μl

-          part 6.3 A C1 and part 6.3 B C2 RD digests incubated at 37 °C for 1 h (then dephosphorylation)

-          RBSrev + pSB1C3 RD digest incubated at 37 °C for 2 h (no dephosphorylation, since Promoter hybridization oligos are dephosphorylated!)

AP treatment of part 6.3 A C1 and B C2

-          40 μl reaction + 5 μl 10x AP buffer + 2 μl AP + 3 μl dH2O

-          incubation for ca. 50 min at 37 °C (although only 15 min needed, since XbaI and EcoRI generate 5’ overhangs. But dephosphorylation often incomplete…)

Gel run of digested and AP treated part 6.3 A C1 and BC2 and RBSrev digested with SpeI and EcoRI

-          1%-agarose-1xTAE gel

-          loading of 3 μl  2 log ladder

-          loading of 4 μl of dephosphorylated reactions + 1 μl 5xLD

-          loading of 3 μl RBSrev digest + 1 μl dH2O + 1μl 5xLD

-          run at 100 V

-          EtBr staining + destaining in water

-          UV detection

Loading:

Marker/ uncut RBSrev C1/RD RBSrev C1/uncut part6.3 A C1/RD + AP part6.3 A C1/uncut part6.3 B C2/RD + AP part6.3 B C2/Marker

-          no over-digest; digests still incomplete, but since AP treatment of at least part 6.3 A and B vector, this should not matter; in case of RBSrev ligation, re-ligands could occur

Purification of digested and dephosphorylated samples

-          with Qiagen PCR purification kit

-          addition of 500 μl PB buffer to each sample

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

-          NanoDrop concentration measurement

Ligation

Ligation of

a) DarRrev insert + Termrev vector →  Termrev-DarRrev in pSB1C3

b) RBSrev hybridization oligo + part6.3 A/part 6.3 B →  part 6.4 A/part 6.4 B (RBSrev-Promoter1/3rev in part 6.2)

c) Promoter1/3rev + pSB1C3 derived from RBSrev in pSB1C3 digest →  Promoter1/3rev in pSB1C3

Pipetting scheme

a) DarRrev insert + Termrev vector →  Termrev-DarRrev in pSB1C3

Uni D’dorf ligation calculator: for a ratio vector:insert = 1:3, one has to use 50 ng of a 2200 bp (= 2070 of pSB1C3 + 130 bp of terminator) vector and 44 ng insert of 650 bp →  approximate amounts used in this ligation.

b) RBSrev hybridization oligo + part6.3 A/part 6.3 B →  part 6.4 A/part 6.4 B (RBSrev-Promoter1/3rev in part 6.2)

c) Promoter1/3rev + pSB1C3 derived from RBSrev in pSB1C3 digest →  Promoter1/3rev in pSB1C3

-          All reaction were pipetted individually/no mastermix

-          Incubation ON at 16 °C (cold room heat block)

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