BioMicroCenter:RTPCR

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(Fluidigm BioMark)
Current revision (23:39, 5 February 2013) (view source)
(Traditional Real Time PCR)
 
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{{BioMicroCenter}}
{{BioMicroCenter}}
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== [[BioMicroCenter:Wafergen | Wafergen Smartchip]] ==
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[[Image:BioMicroCenter-Smartchip.jpg|300px|right]]
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The Wafergen Smartchip system is a highly parallelized system allowing for running thousands of RT-PCR reactions on a single cartridge. Useful for genotyping and gene expression, the Smartchip works using very small reaction volumes (100nL) pipetted with a specialized automated liquid handler. The 72x72 chip can be filled in a broad variety of patterns. RT-PCR in the Wafergen system is compatible with TaqMan(TM), the Roche Universal priming kit, and SYBR Green technologies.
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The Wafergen Smartchip system is currently on loan within the BioMicro Center. Future availability will be based on usage level.
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* <B>[[BioMicroCenter:Wafer_CAL| Schedule time on the Wafergen]]</B><BR><BR>
== [[BioMicroCenter:BioMark | Fluidigm BioMark]] ==
== [[BioMicroCenter:BioMark | Fluidigm BioMark]] ==
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The Fluidigm BioMark system is a highly parallelized system allowing for running thousands of RT-PCR reactions on a single cartridge. Useful for genotyping, gene expression analysis and digital PCR, the BioMark cassette works using thousands of [http://www.fluidigm.com/glossary/gloss_nflex.htm NanoFlex(TM) valves] to fill thousands of individual reactions from a matrix of samples and primers. Standard Geneotyping / Gene Expression cassettes come in 48x48 and 96x96 format allowing either ~2,500 or ~10,000 reactions on a single cassette. RT-PCR in the BioMark system is compatible with TaqMan(TM), the Roche Universal priming kit, and SYBR Green technologies.
The Fluidigm BioMark system is a highly parallelized system allowing for running thousands of RT-PCR reactions on a single cartridge. Useful for genotyping, gene expression analysis and digital PCR, the BioMark cassette works using thousands of [http://www.fluidigm.com/glossary/gloss_nflex.htm NanoFlex(TM) valves] to fill thousands of individual reactions from a matrix of samples and primers. Standard Geneotyping / Gene Expression cassettes come in 48x48 and 96x96 format allowing either ~2,500 or ~10,000 reactions on a single cassette. RT-PCR in the BioMark system is compatible with TaqMan(TM), the Roche Universal priming kit, and SYBR Green technologies.
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Using the BioMark system requires some additional processing over traditional RT-PCR. Using only 10nL(?) in each reaction, ensuring that each reaction has sufficient template is a primary concern. To create a concentrated enough template, the BioMark system requires a pre-amplification where all of the primer pairs to be tested are mixed together with each sample and amplified by standard PCR. This increases the concentration of the specific product enough to ensure that sufficient template is added to each of the final reactions.  
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Using the BioMark system requires some additional processing over traditional RT-PCR. Using only 10nL in each reaction, ensuring that each reaction has sufficient template is a primary concern. To create a concentrated enough template, the BioMark system requires a pre-amplification where all of the primer pairs to be tested are mixed together with each sample and amplified by standard PCR. This increases the concentration of the specific product enough to ensure that sufficient template is added to each of the final reactions.  
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The BioMark system is currently being installed in the BioMicro Center and a calandering system will be added shortly. Currently use of the BioMark costs '''$100''' per cassette to help defray the service contract charge.
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* <B>[[BioMicroCenter:Fluid_CAL| Schedule time on the Biomark]]</B><BR><BR>
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Use of the BioMark costs '''$150''' per use to help defray the service contract charge.
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For training please email biomicro@mit.edu
== Traditional Real Time PCR ==
== Traditional Real Time PCR ==
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[[Image:Opticon2_system.gif|right]]   
 
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===MJ Research Real Time PCR Machine===
 
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The MJ Research PCR Machine enables multicolor capability allowing detection of SYBR® Green I or FAM in the first channel, and a range of fluorophores in the second channel — including TET, HEX, VIC, and TAMRA — for a multitude of applications such as RT-qPCR and allelic discrimination. The DNA Engine® thermal cycler offers precision thermal control and a temperature gradient feature permits simultaneous incubation at 12 different temperatures to optimize reactions in a single run. The real-time results allow for plotting of signal intensity vs. cycle number and graphically monitoring the thermal profile during the run. The extraordinary sensitivity permits reliable detection of one initial template copy, while delivering a linear range of up to ten orders of magnitude in starting copy number (with appropriate chemistry). Users are currently charged '''$10''' per plate for the use of this machine. <BR>
 
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* <B>[[BioMicroCenter:MJO_CAL|Schedule Time on the MJ Opticon]]</B><BR><BR>
 
[[Image:LC480.jpg|right]]
[[Image:LC480.jpg|right]]
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96 well plates ($6 per plate) and SYBR Green ($40 per mL) can also be supplied on demand for use on the LightCycler480
96 well plates ($6 per plate) and SYBR Green ($40 per mL) can also be supplied on demand for use on the LightCycler480
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'''Please Note:''' There are two LightCycler systems (Thing 1 and Thing 2) that have a sign up maximum of four hours per day during business hours (10AM to 5PM), all time slots are available the night before even if it exceeds the maximum. New users who wished to be trained must be [[BioMicroCenter:CoreDeps|Core Lab members]].
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'''Please Note:''' There are three LightCycler systems (Thing 1, Thing 2, and Thing 3) that have a sign up maximum of four hours per day during business hours (10AM to 5PM), all time slots are available the night before even if it exceeds the maximum. New users who wished to be trained must be [[BioMicroCenter:CoreDeps|Core Lab members]].
[http://www.google.com/calendar Schedule Time on the LightCycler 480]
[http://www.google.com/calendar Schedule Time on the LightCycler 480]
* Username: Building68
* Username: Building68
* Password: DNARNAprotein
* Password: DNARNAprotein
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[[Image:Block Changing Protocol LightCycler480.txt]]
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== PCR ==
 
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[[Image:Primus_multiblock.jpg|right]]
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[[Image:Opticon2_system.gif|right]]  
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===MWG Thermal Cyclers===
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===MJ Research Real Time PCR Machine===
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The Primus Multiblock system provides comprehensive state-of-the-art PCR technology.
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They are ideal instruments for high throughput PCR and sequencing. The cyclers allow for
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exchangeable blocks (384/96 well format), motorized lid, HTR Peltier technology for
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fastest heating / cooling rates and optimal results. Patented HPL (High Pressure Lid)
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technology for 100% evaporation prevention.
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The MWG PCR machines are available for non-BMC labs at a $10 charge. If you are interested in the machines you must coordinate use with BioMicro Center staff as the machines are used extensively in many protocols run by the staff.
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''' THE MJ IS CURRENTLY RETIRED WHILE THE WAFERGEN SYSTEM IS IN PLACE '''
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The MJ Research PCR Machine enables multicolor capability allowing detection of SYBR® Green I or FAM in the first channel, and a range of fluorophores in the second channel — including TET, HEX, VIC, and TAMRA — for a multitude of applications such as RT-qPCR and allelic discrimination. The DNA Engine® thermal cycler offers precision thermal control and a temperature gradient feature permits simultaneous incubation at 12 different temperatures to optimize reactions in a single run. The real-time results allow for plotting of signal intensity vs. cycle number and graphically monitoring the thermal profile during the run. The extraordinary sensitivity permits reliable detection of one initial template copy, while delivering a linear range of up to ten orders of magnitude in starting copy number (with appropriate chemistry). Users are currently charged '''$10''' per plate for the use of this machine. <BR>
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* <B>[[BioMicroCenter:MJO_CAL|Schedule Time on the MJ Opticon]]</B><BR><BR>

Current revision

Image:BioMicroCenter-header6.jpg

Contents

Wafergen Smartchip

The Wafergen Smartchip system is a highly parallelized system allowing for running thousands of RT-PCR reactions on a single cartridge. Useful for genotyping and gene expression, the Smartchip works using very small reaction volumes (100nL) pipetted with a specialized automated liquid handler. The 72x72 chip can be filled in a broad variety of patterns. RT-PCR in the Wafergen system is compatible with TaqMan(TM), the Roche Universal priming kit, and SYBR Green technologies.

The Wafergen Smartchip system is currently on loan within the BioMicro Center. Future availability will be based on usage level.

Fluidigm BioMark

The Fluidigm BioMark system is a highly parallelized system allowing for running thousands of RT-PCR reactions on a single cartridge. Useful for genotyping, gene expression analysis and digital PCR, the BioMark cassette works using thousands of NanoFlex(TM) valves to fill thousands of individual reactions from a matrix of samples and primers. Standard Geneotyping / Gene Expression cassettes come in 48x48 and 96x96 format allowing either ~2,500 or ~10,000 reactions on a single cassette. RT-PCR in the BioMark system is compatible with TaqMan(TM), the Roche Universal priming kit, and SYBR Green technologies.

Using the BioMark system requires some additional processing over traditional RT-PCR. Using only 10nL in each reaction, ensuring that each reaction has sufficient template is a primary concern. To create a concentrated enough template, the BioMark system requires a pre-amplification where all of the primer pairs to be tested are mixed together with each sample and amplified by standard PCR. This increases the concentration of the specific product enough to ensure that sufficient template is added to each of the final reactions.

Use of the BioMark costs $150 per use to help defray the service contract charge. For training please email biomicro@mit.edu

Traditional Real Time PCR

Light Cycler 480 II Real-time PCR Machines

The LightCycler 480 Real-Time PCR System is a fully integrated multiwell-plate based real-time PCR platform for highly accurate qualitative and quantitative detection of nucleic acids. Building on the benefits of Roche's capillary-based LightCycler® Systems, it goes one step further in offering enhanced throughput, compatibility with automation equipment and maximum flexibility regarding hard- and software. Providing novel ways to combine speed and accuracy without compromises, the LightCycler® 480 Real-Time PCR System meets the needs of a broad range of applications in research fields such as gene expression studies, discovery and analysis of genetic variation or array data validation.

There is a $10 charge per plate will be implemented to cover the cost of the service contract. The LightCycler 480 is only available for use by MIT Core departments.

96 well plates ($6 per plate) and SYBR Green ($40 per mL) can also be supplied on demand for use on the LightCycler480

Please Note: There are three LightCycler systems (Thing 1, Thing 2, and Thing 3) that have a sign up maximum of four hours per day during business hours (10AM to 5PM), all time slots are available the night before even if it exceeds the maximum. New users who wished to be trained must be Core Lab members.

Schedule Time on the LightCycler 480

  • Username: Building68
  • Password: DNARNAprotein

Image:Block Changing Protocol LightCycler480.txt


MJ Research Real Time PCR Machine

THE MJ IS CURRENTLY RETIRED WHILE THE WAFERGEN SYSTEM IS IN PLACE The MJ Research PCR Machine enables multicolor capability allowing detection of SYBR® Green I or FAM in the first channel, and a range of fluorophores in the second channel — including TET, HEX, VIC, and TAMRA — for a multitude of applications such as RT-qPCR and allelic discrimination. The DNA Engine® thermal cycler offers precision thermal control and a temperature gradient feature permits simultaneous incubation at 12 different temperatures to optimize reactions in a single run. The real-time results allow for plotting of signal intensity vs. cycle number and graphically monitoring the thermal profile during the run. The extraordinary sensitivity permits reliable detection of one initial template copy, while delivering a linear range of up to ten orders of magnitude in starting copy number (with appropriate chemistry). Users are currently charged $10 per plate for the use of this machine.

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