Team:Goettingen/NoteBook w12
From 2013.igem.org
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Gel extraction of DarRrev insert , Restriction digest of Termrev with PstI FD, Restriction digest of RBSrev C1 and part6.3 A and B (C2 each) with EcoRI FD, AP treatment of Termrev vector for ligation with DarRrev insert , Restriction Digest of all vectors digested with EcoRI FD today, ligation.....
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
sample | ng/μl | A260nm/A280nm | A260nm/A230nm |
DarRrev insert X+P | 14.8 | 1.53 | 0.12 |
Restriction digest of Termrev with PstI FD
- DNA: Termrev C1 plasmid restricted with SpeI and purified yesterday
- digest:
Component | Volume |
plasmid | 30μl (volume determined with pipet) |
10x FD Green buffer | 4μl |
PstI FD | 4μl |
dH2O | 2μl |
Total | 40μl |
- 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):
Component | Volume |
plasmid | 8μl (since each plasmid had > 200 ng/μl) |
10x FD Green buffer | 4μl |
Eco I FD | 4μl |
dH2O | 24μl |
Total | 40μ |
- 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
![](https://static.igem.org/mediawiki/2013/b/b3/Goe-20.08.13-RT-1.png)
- 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:
| Primers | approx. PCR product size (w/o prefix/suffix) | approx. PCR product size (w/ prefix/suffix) |
Riboswitch with native Promoter and RBS | iGEM_42 iGEM_43 | 460 bp | 500 bp |
Riboswitch with native Promoter | iGEM_42 iGEM_41 | 315 bp | 355 bp |
Riboswitch only | iGEM_40 iGEM_41 | 230 bp | 270 bp |
Riboswitch with native RBS | iGEM_40 iGEM_43 | 375 bp | 415 bp |
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
![](https://static.igem.org/mediawiki/2013/7/79/Goe-20.08.13-RT-2.png)
- 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
![](https://static.igem.org/mediawiki/2013/d/d4/Goe-20.08.13-RT-3.png)
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.
Component | With DarRrev insert | Without insert control |
Termrev vector (purified today, 19.9 ng/µl) | 3 µl | 3 µl |
DarRrev insert (purified today, 14.8 ng/µl) or dH2O for w/o insert control | 3 µl | 3 µl |
T4 ligase (ThermoScientific) | 1 µl | 1 µl |
10x T4 ligase buffer (ThermoScientific) | 1 µl | 1 µl |
dH2O | 2 µl | 2 µl |
Total | 10 µl | 10 µl |
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)
Component | With RBSrev insert | Without insert control |
Part 6.3 A vector (purified today, 30.5 ng/µl) | 2 µl | 2 µl |
RBSrev hybridization oligo (prepared on 19.8.13) or dH2O for w/o insert control | 8 µl | 8 µl |
T4 ligase (ThermoScientific) | 2 µl | 2 µl |
10x T4 ligase buffer (ThermoScientific) | 2 µl | 2 µl |
dH2O | 6 µl | 6 µl |
Total | 20 µl | 20 µl |
Component | With RBSrev insert | Without insert control |
Part 6.3 B vector (purified today, 33.6 ng/µl) | 2 µl | 2 µl |
RBSrev hybridization oligo (prepared on 19.8.13) or dH2O for w/o insert control | 8 µl | 8 µl |
T4 ligase (ThermoScientific) | 2 µl | 2 µl |
10x T4 ligase buffer (ThermoScientific) | 2 µl | 2 µl |
dH2O | 6 µl | 6 µl |
Total | 20 µl | 20 µl |
c) Promoter1/3rev + pSB1C3 derived from RBSrev in pSB1C3 digest → Promoter1/3rev in pSB1C3
Component | With Promoter1rev insert | With Promoter3rev insert | Without insert control |
psB1C3 vector (derived from RBSrev C1 plasmid, purified today, 29.9 ng/µl) | 2 µl | 2 µl | 2 µl |
Promoter1/3rev hybridization oligo (prepared on 19.8.13) or dH2O for w/o insert control | 10 µl | 10 µl | 10 µl |
T4 ligase (ThermoScientific) | 2 µl | 2 µl | 2 µl |
10x T4 ligase buffer (ThermoScientific) | 2 µl | 2 µl | 2 µl |
dH2O | 4 µl | 4 µl | 4 µl |
Total | 20 µl | 20 µl | 20 µl |
- All reaction were pipetted individually/no mastermix
- Incubation ON at 16 °C (cold room heat block)
![](https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png)
Mini-Preps of RiboA Clones 1, 2, 4 and 5 (see gel from 12.08.), RD of Ribo 2,4,5, Ligations of the Riboswitches into next vectors, MiniPrep of RBSrev + pSB1C3, Prom1rev + part6.2, Prom3rev + part 6.2 (C1 – C3 each) and PCR test, PCR to test, if RBSrev C1 – C3 are positive or negative, Test restriction digest of Prom1rev + part6.2, Prom3rev + part 6.2 (C1 – C3 each)....
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:
Sample | ng/µl | A260nm/A280nm | A260nm/A230nm |
Ribo A clone 1 | 213.9 | 1.89 | 1.86 |
Ribo A clone 2 | 152.5 | 1.95 | 2.00 |
Ribo A clone 4 | 396.6 | 1.90 | 2.11 |
Ribo A clone 5 | 429.4 | 1.89 | 2.18 |
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
![](https://static.igem.org/mediawiki/2013/c/c2/Goe-19.08.13-RT-1.png)
→ 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):
Component | Volume |
Taq | 1 μl |
10x Taq buffer | 2.5 μl |
dNTP mix | 1 μl |
iGEM_38 (VF2) 1:20 | 1 μl |
iGEM_39 (VR) 1:20 | 1 μl |
dH2O | 17.5 μl |
template | 1 μl |
Total | 25 μl |
- 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:
Component | Volum |
Taq | 6μl |
10x Taq buffe | 15μl |
dNTP mix | 6μl |
iGEM_38 (V2) 1:20 | 6μl |
iGEM_39 (VR) 1:20 | 6μl |
dH2O | 105μl |
Total | 144μl |
→ 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
Component | Volume |
plasmid | 2μl |
10x FD Green buffer | 1μl |
EcoRI FD | 1μ |
PstI FD | 1μl |
dH2O | 5μl |
Total | 10μl |
- the plasmid was added to the tubes, then addition of 8 μl MasterMix; the MasterMix consisted of…
Component | Volume |
10x FD Green buffer | 7μl |
EcoRI FD | 7μl |
PstI FD | 7μl |
dH2O | 35μl |
Total | 56μl |
- 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
![](https://static.igem.org/mediawiki/2013/1/1d/Goe-19.08.13-RT-2.png)
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
![](https://static.igem.org/mediawiki/2013/0/05/Goe-19.08.13-RT-3.png)
- 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
![](https://static.igem.org/mediawiki/2013/3/3e/Goe-19.08.13-RT-4.png)
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:
![](https://static.igem.org/mediawiki/2013/e/e1/Goe-19.08.13-RT-5.png)
- a bit was lost, but that’s ok…
- gel pieces weighed and stored in DarR reporter system box at – 20 °C
![](https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png)
Riboswitch A Clones 1, 2, 4 and 5 inocculated into 4ml Cm cultures for minipreps tomorrow