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Team:Goettingen/NoteBook w10
From 2013.igem.org
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
