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Team:Warsaw/Calendar-Main/8 July 2009
From 2009.igem.org
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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
Cloning the p53 coding sequence
Marcin
Comment
There were some difficulties with ligation p53 coding sequence into pKS plasmid so I decided to take another sample containing isolate amplified p53 (via PCR reaction) and perform digest of the sample using XbaI
Tasks:
- Restriction digest of p53 coding sequence obtained from PCR reaction
Methods:
- Reaction mixture composition:
22.5 ul PCR product (DNA concentration about 6.5 ng/ul), 2.5 ul Tango Buffer (Fermentas), 0.5 ul XbaI (Fermentas)
- Digest program:
Digest
3h 37 °C 15 min 80 °C ~4 °C
- Quantification of the amount of DNA after digest on the agarose gel
Methods:
- Electrophoresis of the digested DNA sample:
agarose concentration - 1%
voltage - 70V
time - about 30 minutes
After electrophoresis gel was irradiated with UV light and photographed:
Comment
The sample must have degraded after gel-out procedure. Most probably due to acidic condition in the solution DNA hydrolysed. It is obligatory to do another PCR reaction using the old plasmid sample (I hope the DNA is not degraded!).
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
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
Jarek
Task:
- Preparation of bacterial cultures containing parts R0010, B0032 and C0051 in LB with ampicilin
Methods:
- Concentration of ampicilin used for cultures was 100 ug/ml
Results:
- The growth of the cultures will be observed on the next day
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