Addition of 100 ul of T7 phage solution to 100 ml of bacteria with an OD600 of around 0.80
After 4, 8 and 12 minutes bacteria were spun down (50 ml falcon) quickly at max speed (7000 rcf) for 15-30 seconds
Supernatant was discarded
Bacterial pellet was instantly lysed by adding Phenol/Chloroform/Isoamylalcohol
Half of the duplicates were treated with DNase I (NEB) as described in the following:
- Resuspension of the RNA-pellet with 90 ul nf H20
- Addition of 10 ul 10x DNase I buffer
- Addition of 1 ul (2 U) DNase I
0.5 ul cDNA,
5 ul Luna MM,
3.5 ul nf H2O,
(create Master Mix, multiply equivalently)
1 ul 2.5 mM Primer Mix F + R,
(pipet in directly in each well)
From Up to Down: T7p29 TP01 recC rzlys hcaT idnT rrsA cysG
Results:
no results
Test of Primers at Different Annealing Temperatures
150 ul of an ON-Culture of T7 Bacteria were pipetted to 5 ml of LB in 10 different tubes
At OD=0.8; 5 ul / 2,5 * 10^7 T7 Phages (5*10^9 PFU/ml) were added to each tube
After 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 minutes the cells were briefly spun down (accelerate to 7000 rcf and then decelerate). Start centrifugation 1 minute and 15 seconds before the timepoint to freeze on time.
Discard supernatant and immerse for 10 seconds in liquid N2
Store pellet at -80°C
Lyse frozen pellet. Follow Promega SV Total RNA Isolation Kit Instructions (exept: 500 ul Lysis Buffer, 100 ul DNase solution per column, 20 min DNase incubation)
Results:
Did not work, Nanodrop shows no RNA
PCR Primer Testing- PCR for CysG, idnT, rrsA, hcaT
150 ul of an ON-Culture of T7 Bacteria were pipetted to 5 ml of LB in 12 different tubes (10 Timepoints + Timepoint 0 + Reference for OD) Note: 150 ul ON-Bacteria added at 13:00, OD at 0.12 after 20 mins; OD at 0.47 after 40 mins
At OD=0.8; 5 ul / 2,5 * 10^7 T7 Phages (5*10^9 PFU/ml) were added to each tube
3.5 % Urea gels with Acrylamid:Bisacrylamid 29:1 were prepared.
Run gel at 120 V, 400 mA for 1h20Min.
Stain in TBE Buffer with SybrGreen II for ~30 Minutes
Results:
50S and 30S rRNA are clearly visible, proportion shows RNA integrity
Positive Control: 2 ul Phages (2,5*10^9 pfu/ml)
Negative Control: 2 ul nf H2O
RNA: Pooled RNA from all samples: 2 ul
NEB OneTaq (NEB, M0480); 250 uM Primer (TP01 F+R)
420 ng RNA in 21.4 ul water (see table from 6.8)
25mM MgCl2: 8 ul
10 x Buffer: 4 ul
10 mM dNTP: 4 ul
RNasin: 1 ul
AMV RT: 0.6 ul
Reverse Primers: 1ul 10 mM Mix (final 250 nM)
Pipet 1 ul 2.5 uM Primer directly into well
Create Master Mix: 1 ul cDNA, 3 ul nf H2O, 5 ul Luna qPCR Master Mix
Pipet 9 ul of MM into well, spin down thouroughly!
Results:
rrsA for 0 min is not correct; line 15 and 12 inverted!
