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Team:Warsaw/Calendar-Main/8 July 2009
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Gradient PCR Pho
Kama
Kama
Tasks:
- Amplification of phoP/phoQ
Methods:
PCR mixture's composition:
1ul pfu buffer (Fermentas), 1ul MgSO4 (Fermentas), 0,5ul primers, 0,5ul dNTPs (10 mM), 0,25ul pfu turbo polymerase, 0,5ul template DNA from Listeria, optionally: 0,75ul DMSO, solution was topped up with H2O to 10ul.
- PCR programs:
pho
4min 95°C
(30s 95°C, 1min 45-55°C, 4min 72°C)x3
(30s 95°C, 1min 55-60°C, 4min 72°C)x28
10min 72°C
~ 7°C
- Electrophoretic separation on 1% agarose gel
Results:
- Gel (from left)
- control -
- 2-6 - samples (annealing temperature increases to from the left to the right)
- 7-11 - samples with DMSO (annealing temperature increases to from the left to the right)
- M - GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
Notes:
Isolation and digest of pKS-CRO (ligated and transformed into E. coli the previous day)
Kuba
- pKS-CRO cut with XbaI and EcoRI
.JPG)
- Gel (from the left)
- GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
- uncut plasmid (isolate no.1)
- cut plasmid (isolate no.1)
- uncut plasmid (isolate no.2)
- cut plasmid (isolate no.2)
- uncut plasmid (isolate no.3)
- cut plasmid (isolate no.3)
Note: isolate no.1 contains a correctly inserted PCR product
Miecznikowa team Aim: debug RFP-terminator ligation that did not work
Jarek/Franek/Ania
Jarek/Franek/Ania
Task1:
- Alkaline lysis of the plasmid containing Terminator
Methods:
PlasmidMini set by A&A Biotechnology was used. 2 test tubes with 5ml LB and 5 ul Ampicyline were first inoculated with the plasmid containing colonies. The cultures were incubated over night at 37C.Alkaline lysis was performed on both cultures. The pellet from 5 ml of bacteria was used.
Results:
- DNA extraction quality controll.DNA Concentration was measured using Spectrophotometer NanoDrop ND-1000
| DNA sample | DNA concentration in ng |
|---|---|
| Terminator sample 1 (Ter1) | 73.9 ng/ul |
| Terminator sample 1 (Ter2) | 85.46 ng/ul |
Task2:
- We decided to measure the concentration of DNA in our samples for future use e.g. efficient ligation mix. NanoDrop ND-1000 was used.
Results:
| DNA sample | DNA concentration in ng |
|---|---|
| Rfp3 1st measurement | 17.49 ng/ul |
| Rfp3 2nd | 24.18 ng/ul |
| Rfp3 3rd | 24.50 ng/ul |
| Rfp4 1st measurement | 28.85 ng/ul |
| Rfptra (digested plasmid with RFP) | 0.4 ng/ul |
| Rfptra 2nd measurement | 0.8 ng/ul |
| RBS digested (digested RBS) | 1.44 ng/ul |
| RBS digested 2nd measurement | 0.72 ng/ul |
Notes - IMPORTANT:
- Rfptra and RBSdigested samples were extracted from the gel using gel-out kit by A&ABiotechnology. Presented data shows that after gel extraction there is very little DNA in the sample.
Probably there is a ****problem with gel extraction procedure**** - the Gel-out set by A&A Biotechnology might be defective.
Miecznikowa team - division of labour;)
Today the intermediate bricks were designed and we divided the tasks as follows:
- Franek: BBa_J5528, digestion with (EcoRV, PstI) and insertion into pKS2 (Bluescript) cut with SmaI i PstI (look for white colonies on the X-gal, IPTG, Amp plates)
- Jarek: construct BBa_K177011
- Michał: construct BBa_K177012
- Marek: construct BBa_K77013
- Ania: construct BBa_K177014
- Monika: transform competent cells with BBa_1763004, BBa_S03473, BBa_E0840, BBa_J07037 out of the distribution.
potential problems: get inv/llo/phoP/phoQ
Miecznikowa team: adjusting part BBa_J5528 to Bba_K177011
Franek
Task:
- transform competent cells with BBa_I0500
Methods:
- Resuspension of DNA from plate 1, 14N (BBa_I0500) with 15ul of H2O
- Transformation of chemocompetent cells with 4ul of BBa_I0500 DNA solution
- Plating bacterias on LB medium supplemented with kanamycin
Results:
- Will be determined tomorrow
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