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Team:METU Turkey/Delta
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| + | <br> PROTEIN-DNA INTERACTIONS | ||
| + | |||
| + | <br> Aim: | ||
| + | <br> To show the interaction thermodynamics of COOA protein and target promoter pCooF and pCooM and changes in binding affinity of protein with respect to mutational changes in its target DNA. | ||
| + | |||
| + | <br> Methods | ||
| + | <br> - Isothermal Titration Calorimetry (ITC) | ||
| + | <br> - Electrophoretic Mobility Shift Assay (EMSA) | ||
| + | <br > - Intrinsic Tryptophan Fluorescence (ITF) | ||
| + | |||
| + | <br>Both these methods require short oligonucleotides as DNA template and especially for ITC experiment we need high amounts of DNA template. In order to propagate enough DNA, we design primers for promoters and sequences, which include response elements. | ||
| + | <br> 1.Isothermal Titration Calorimetry (ITC) | ||
| + | |||
| + | <br> Scenarios for DNA component preparation: | ||
| + | |||
| + | <br> 30bp | ||
| + | <br> The possible sequence of oligonucleotide that we are planning to use in ITC measurements: | ||
| + | GGA TAA CTG TCA TCT GGC CGA CAG ACG GGG | ||
| + | Response (RE) Response (RE) | ||
| + | Element Element | ||
| + | |||
| + | <br>- MW : 18594.2 g/mole | ||
| + | <br> - GC content: 60% | ||
| + | <br>- Length: 30bp | ||
| + | <br>- Average MW of a base is 325 g/mole (our MW prediction is applicable acc to that info.) | ||
| + | <br> - for an ITC experiment min 1uM DNA ( max: 100uM) is required in sample cell (Vcell 1.8mL) | ||
| + | <br> - we will prepare 2.5 mL per experiment ( it is calculated as 2.5 nmole - 18kDa x 2.5nmole= 45ug) | ||
| + | <br> - for 1uM DNA we require 45ug DNA in 2.5 mL | ||
| + | |||
| + | <br> If we propagate our oligonucleotide w/ PCR: | ||
| + | <br> - min 70 bp is required for using a QIAGEN PCR purification kit >>>it is problem for us | ||
| + | <br> - max yield is 10ug | ||
| + | <br> - so we will use 5 kit per experiment for getting 45ug | ||
| + | |||
| + | |||
| + | |||
| + | <br>50bp | ||
| + | <br> TAA AAA CTC TGG ATA ACT GTC ATC TGG CCG ACA GAC GGG GGC CGG GCT TT | ||
| + | <br> - Length: 50bp | ||
| + | <br> - MW: 30773g/mole ~31 kDa | ||
| + | <br> - for ITC 1uM (Vcell = 2.5 mL) | ||
| + | <br> 1umol in 1 L ; so we need 2.5nmole for 2.5 mL | ||
| + | <br>1 mole is nearly 31000 g ; so we need 77.7 ug - w/ invitrogen plasmid purification kit we get 800ug plasmids from 100ml culture - we can extract 800ug plasmid from 2ml culture - if we assume that our vector plasmid is nearly 5000bp long, it will be 100 fold of our fragment DNA (50bp) - 800/100 we get 8ug fragments after restriction digestion. | ||
| + | <br> - w/ qiagen gel extraction kit we can get 10ug DNA once | ||
| + | <br> - so, we need 8 kit gel extraction and 10 plasmid purification kit for one ITC experiment | ||
| + | <br> - 50bp fragment recovery 50% (Fermentas) | ||
| + | <br> - for 50 bp we require ~80ug | ||
| + | <br> - one kit elutes 25x 0.05= 12.5ug in once | ||
| + | <br> - for 80ug we require 80/12.5= 6.4= 7 adet kit | ||
Revision as of 01:19, 28 October 2010
PROTEIN-DNA INTERACTIONS
Aim:
To show the interaction thermodynamics of COOA protein and target promoter pCooF and pCooM and changes in binding affinity of protein with respect to mutational changes in its target DNA.
Methods
- Isothermal Titration Calorimetry (ITC)
- Electrophoretic Mobility Shift Assay (EMSA)
- Intrinsic Tryptophan Fluorescence (ITF)
Both these methods require short oligonucleotides as DNA template and especially for ITC experiment we need high amounts of DNA template. In order to propagate enough DNA, we design primers for promoters and sequences, which include response elements.
1.Isothermal Titration Calorimetry (ITC)
Scenarios for DNA component preparation:
30bp
The possible sequence of oligonucleotide that we are planning to use in ITC measurements: GGA TAA CTG TCA TCT GGC CGA CAG ACG GGG Response (RE) Response (RE) Element Element
- MW : 18594.2 g/mole
- GC content: 60%
- Length: 30bp
- Average MW of a base is 325 g/mole (our MW prediction is applicable acc to that info.)
- for an ITC experiment min 1uM DNA ( max: 100uM) is required in sample cell (Vcell 1.8mL)
- we will prepare 2.5 mL per experiment ( it is calculated as 2.5 nmole - 18kDa x 2.5nmole= 45ug)
- for 1uM DNA we require 45ug DNA in 2.5 mL
If we propagate our oligonucleotide w/ PCR:
- min 70 bp is required for using a QIAGEN PCR purification kit >>>it is problem for us
- max yield is 10ug
- so we will use 5 kit per experiment for getting 45ug
50bp
TAA AAA CTC TGG ATA ACT GTC ATC TGG CCG ACA GAC GGG GGC CGG GCT TT
- Length: 50bp
- MW: 30773g/mole ~31 kDa
- for ITC 1uM (Vcell = 2.5 mL)
1umol in 1 L ; so we need 2.5nmole for 2.5 mL
1 mole is nearly 31000 g ; so we need 77.7 ug - w/ invitrogen plasmid purification kit we get 800ug plasmids from 100ml culture - we can extract 800ug plasmid from 2ml culture - if we assume that our vector plasmid is nearly 5000bp long, it will be 100 fold of our fragment DNA (50bp) - 800/100 we get 8ug fragments after restriction digestion.
- w/ qiagen gel extraction kit we can get 10ug DNA once
- so, we need 8 kit gel extraction and 10 plasmid purification kit for one ITC experiment
- 50bp fragment recovery 50% (Fermentas)
- for 50 bp we require ~80ug
- one kit elutes 25x 0.05= 12.5ug in once
- for 80ug we require 80/12.5= 6.4= 7 adet kit
