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Team:Goettingen/NoteBook w10
From 2013.igem.org
PCR for amplification of different riboswitch inserts: Test PCR for optimal primer concentration, Gel run: Riboswitch Test PCR and Test RD of plasmids from YFP and CFP clones, Primers for generating reverse terminator insert
PCR for amplification of different riboswitch inserts: Test PCR for optimal primer concentration
- Primers iGEM_67/68/69/70
1x reaction (50 µl)
Component | Volume Negative control | Volume 1x primer concentration | Volume 2x primer concentration | Volume 4x primer concentration |
5x HF buffer | 10 µl | 10 µl | 10 µl | 10 µl |
dNTP mix (12.5 mM each) | 2 µl | 2 µl | 2 µl | 2 µl |
Primer fwd (5 pmol) | 2 µl | 2 µl | 4 µl | 8 µl |
Primer rev (5 pmol) | 2 µl | 2 µl | 4 µl | 8 µl |
Chromosomal DNA B. subtilis 168 (or water for neg. control) | 1 µl | 1 µl | 1 µl | 1 µl |
PfuS | 1 µl | 1 µl | 1 µl | 1 µl |
dH2O | 32 µl (12 µl) | 32 µl (12 µl) | 28 µl (8 µl) | 20 µl (0 µl) |
- preparation of master mix for 20 reactions containing
Component | Volume |
dNTP mix (12.5 mM each) | 40 µl |
5x HF buffer | 200 µl |
PfuS | 20 µl |
dH2O | 400 µl |
Total | 660 µl |
- addition of template (chrom. DNA/dH2O), primers and a part of the water (indicated in parenthesis in table for composition of each reaction) individually, then addition of 33 µl master mix
Step | Temperature | Time |
Initial denaturation | 98.5 °C | 5 min |
Denaturation | 98.5 °C | 30 s |
Annealing | Different; see table below… (TA = TM (≈66 °C) – 6 °C) | 35 s |
Elongation | 72 °C | 2 min |
Final elongation | 72 °C | 10 min |
Hold | 15 °C | ∞ |
| Primers | TA = 0.5 * [Tm(primer1) + Tm(Primer2)] – 6 °C | insert size (no overhangs from primers) | Name of Protocol in cycler | Colour code of epi tube |
Riboswitch with native Promoter and RBS | iGEM_69 iGEM_70 | 51 °C
| 466 bp | Ribo1 | Orange |
Riboswitch with native Promoter | iGEM_69 iGEM_68 | 55.2°C | 370 bp | Ribo2 | Violett |
Riboswitch only | iGEM_67 iGEM_68 | 59.7°C | 213 bp | Ribo3 | Yellow |
Riboswitch with native RBS | iGEM_70 iGEM_67 | 55.5 °C | 403 bp | Ribo4 | Blue |
Gel run: Riboswitch Test PCR and Test RD of plasmids from YFP and CFP clones
- 1.5 % Agarose-1xTAE gel
- 4 µl PCR reaction + 1 µl 5x DNA Loading Dye
- 5 µl of Test RDs; 1 µl uncut plasmid + 1 µl 5x DNA Loading Dye + 3 µl dH₂O
- 3 µl 2 log ladder
- Run at ca. 100 V in 1xTAE buffer
- Staining in EtBr and destaining in water
- UV detection
Gel 1:
Gel 2:
-> RD: Expected band ca. 750 bp + ca. 2 kb for all digests. The expected bands were obtained for all RDs --> all clones contain plasmids --> prepare the plasmids for sequencing by G2L
-> PCR: for expected products: add ca. 2x 20 bp to sizes indicated above. The expected bands were obtained. However, for iGEM_67/68, iGEM_69/68 and iGEM_67/70, a slight band at approx. the bp of 2x expected was observed for 2 µl reaction (“product dimmers?”), this seemed to decrease with increasing primer amount. Hig iGEM_69/70 primer amounts seemed to interfere with PCR. The negative control only shows primer clouds at bottom of lanes. The optimal primer concentrations for the different PCR reactions seem to be:
| Primers | Primer amount (1:20 dilution) |
Riboswitch with native Promoter and RBS | iGEM_69 iGEM_70 | 2 µl |
Riboswitch with native Promoter | iGEM_69 iGEM_68 | 4 µl |
Riboswitch only | iGEM_67 iGEM_68 | 4 µl |
Riboswitch with native RBS | iGEM_70 iGEM_67 | 4 µl |
Primers for generating reverse terminator insert
-> Primers arrived: dissolved in HPLC water and diluted 1:20
-> Primers tend to form secondary structures --> Test PCR for optimal primer concentration
-> Annealing temperature: TA = 0.5 * [Tm(iGEM_71) + Tm(iGEM_72)] – 6 °C = 0.5 *[60 °C + 59.1] – 6 °C =53.55 °C
TEST PCR
1x reaction (50 µl) using part 7 C1 plasmid as template
Plasmid concentration when used as a template: 10 – 20 ng/ reaction
Part 7 C1 purified on 11.7.13; concentration = 120.5 ng/µl --> 1:10 dilution in water --> ca. 12 ng/µl
Component | Volume Negative control | Volume 1x primer concentration | Volume 2x primer concentration | Volume 4x primer concentration |
5x HF buffer | 10 µl | 10 µl | 10 µl | 10 µl |
dNTP mix (12.5 mM each) | 2 µl | 2 µl | 2 µl | 2 µl |
Primer fwd (5 pmol) | 2 µl | 2 µl | 4 µl | 8 µl |
Primer rev (5 pmol) | 2 µl | 2 µl | 4 µl | 8 µl |
Part 7 C1 plasmid (ca. 12 ng/µl) or water for negative control | 1 µl | 1 µl | 1 µl | 1 µl |
PfuS | 1 µl | 1 µl | 1 µl | 1 µl |
dH2O | 32 µl (12 µl) | 32 µl (12 µl) | 28 µl (8 µl) | 20 µl (0 µl) |
- preparation of master mix for 5 reactions containing
Component | Volume |
dNTP mix (12.5 mM each) | 10 µl |
5x HF buffer | 50 µl |
PfuS | 5 µl |
dH2O | 100 µl |
Total | 165 µl |
- addition of template (chrom. DNA/dH2O), primers and a part of the water (indicated in parenthesis in table for composition of each reaction) individually, then addition of 33 µl master mix
PCR protocol
Step | Temperature | Time |
Initial denaturation | 98.5 °C | 5 min |
Denaturation | 98.5 °C | 30 s |
Annealing | 53.5 °C | 35 s |
Elongation | 72 °C | 2 min |
Final elongation | 72 °C | 10 min |
Hold | 15 °C | ∞ |
-> run ON
Restriction digest of DarR (polyadenylated PCR product), Purification of pBluescript(RD with EcoRI) from gel, Ligation of DarR/hybridization oligo inserts with pSB1C3/part6, Gel run of DarR insert and pBluescript+EcoRI, Design of oligos for self-hybridization, Transformation of parts from distribution kit 2013 (CFP, YFP)
Restriction digest of DarR (polyadenylated PCR product)
-> Volume: 12 µl of unpurified DarR polyA reaction
- Preparation of the digest:
DarR polyA reaction mix | 12 µl |
EcoRI FD | 1.5 µl |
PstI FD | 1.5 µl |
10xFD buffer | 2 µl |
dH₂O | 3 µl |
Total | 20 µl |
-> 1 h at 37 °C in ThermoCycler
- Purification of RD reaction using PCR clean up kit (Qiagen): 500 µl PB buffer, 1x elution with 22 µl pre-warmed HPLC water (incubation for ca. 4 min at RT, ca. 1 min at 50 °C)
- Concentration (NanoDrop)
Sample | ng/µl | A260nm/A280nm | A260nm/A230nm |
DarR insert E+P | 6.1 | 1.34 | 1.15 |
Purification of pBluescript(RD with EcoRI) from gel
- 1.1088 g (2 ml epi with gel) – 1.0376 g (another empty 2ml epi) = 0.0712 g --> ca. 70 mg gel --> 210 µl QG buffer were used
- Elution with 2x 22 µl pre-warmed HPLC water
- Concentration (NanoDrop)
Sample | ng/µl | A260nm/A280nm | A260nm/A230nm |
pBluescript + EcoRI | 6.6 | 1.92 | 0.09 |
Ligation of DarR/hybridization oligo inserts with pSB1C3/part6
- Ligation reactions
A) DarR insert (from polyA product) + pSB1C3 E+P (from part 7; 31.7.13)
B) hybridization oligo Promoter 3-DarRoperator + pSB1C3 E+S (from part 7)
C) hybridization oligo Promoter 3-DarRoperator + part 6 E+ X
For DarR ligation: ratio 1:3 was kept --> ca. 50 ng vector (2070 bp) + ca. 43 – 51 ng insert (600-700 bp)
For Hyp oligo: 20- 30 ng vector + half amount of reaction mix from
Component | DarR | Hyb oligo + pSB1C3 | Hyb oligo + part 6 |
Insert/dH₂O (w/o insert control) | 8 µl (6.1 ng/µl) | 8.05 µl | 8.05 µl |
T4 Ligase (ThermoScientific) | 2 µl | 2 µl | 2 µl |
T4 ligation buffer 10x | 2 µl | 2 µl | 2 µl |
Vector | 1 µl (56.6 ng/µl) | 5 µl (6.3 ng/µl) | 3 µl (9.8 ng/µl) |
dH₂O | 7 µl | 4.95 µl | 2.95 µl |
Total | 20 µl | 20 µl | 20 µl |
-> Incubation at 16 °C for several hours (started at 11:30)
Gel run of DarR insert and pBluescript+EcoRI
- 1 % agarose-1xTAE gel
- Loading of 3 µl 2 log ladder
- Loading of 1 µl pBluescript (2.8.13; 342.0 ng/µl) + 3 µl dH₂O + 1 µl 5xLD
- Loading of 4 µl pBluescript purified EcoRI RD + 1 µl 5xLD
- Run at 100 V
- EtBr staining + destaining in water
- UV detection
Gel:
>-> EcoRI-linearzied pBluescript seems to be pure. Still, shortly below the linearized-plasmid-band, some additional weak bands were seen --> possibly unspecific side products? But the strong band of the supercoil-plasmid is gone --> further digest with PstI
-> For DarR insert, an additional band at 0.1 kb was obtained --> polyA’s that were cut off by the enzymes??? Or a non-specific side product from PCR? --> if this fragment can be cloned, we will notice it when the transformants are tested in Colony PCR…
Digest of pBluescript-EcoRI with PstI
Ca. 33 µl pBluescript left
-> Addition of 4 µl FD buffer and 4 µl PstI FD; incubation for 1 h at 37 °C
Design of oligos for self-hybridization
-> These oligos contain the reverse complement sequence of the parts indicated below, since they are designed for reverse integration into pSB1C3 (DarR transcription unit)
-> Oligos hybridization leads to EcoRI and SpeI overhangs
- For strong RBS (part 8)
- For promoters parts 1,2,3,4 (an additional sequence of ca. 60 bp (reverse translation of “cyclicdiampacterim”) was added in front of the promoter so that binding of RNA-Pol does not interfere with binding of RNA-Pol to promoter of GFP transcription unit)
Transformation of parts from distribution kit 2013 (CFP, YFP)
Inoculation of 3 clones of each transformation in LBcm for miniprep
Preparation of back-up plates
-> Forgotten in fridge (4 °C) on 5.8.13
-> Put to 37 °C by Dominik today
