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BioMicroCenter:DNA HTL: Difference between revisions
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The BioMicro Center offers high-throuhgput DNA library preparation for NexteraXT samples and for metagenomics/amplicon generation. High-throughput library generation is different from standard library preparation in a couple key ways. First, with dozens of samples to prepare, small numbers of samples may fail and the cost of reprepping those samples is NOT included in the quoted cost. For some experiments, repreps can be done by hand, but at a higher rate. Second, we are focused far more on reducing price than for standard library preparation, so some "routine services" - such as quality control or arraying of samples - is not built in. | The BioMicro Center offers high-throuhgput DNA library preparation for NexteraXT samples and for metagenomics/amplicon generation. High-throughput library generation is different from standard library preparation in a couple key ways. First, with dozens of samples to prepare, small numbers of samples may fail and the cost of reprepping those samples is NOT included in the quoted cost. For some experiments, repreps can be done by hand, but at a higher rate. Second, we are focused far more on reducing price than for standard library preparation, so some "routine services" - such as quality control or arraying of samples - is not built in. | ||
== High-Throughput Nextera XT == | == High-Throughput and Very-High Throughput Nextera XT == | ||
To minimize library prep costs for standard Illumina libraries, we have automated [[BioMicroCenter:DNA_LIB#NEXTERA_.2F_NEXTERAXT|Illumina NexteraXT]] on our [[BioMicroCenter:Tecan_Freedom_Evo|Tecan EVO150s.]] Libraries will be built to include variable i5 and i7 indexes based on the grid so dual indexing is required during sequencing of these libraries. For more information about how Nextera works, please check out the [[BioMicroCenter:DNA_LIB#NEXTERA_.2F_NEXTERAXT|standard Nextera library preparation section]] of [[BioMicroCenter:DNA_LIB|standard DNA library prep.]] | To minimize library prep costs for standard Illumina libraries, we have automated [[BioMicroCenter:DNA_LIB#NEXTERA_.2F_NEXTERAXT|Illumina NexteraXT]] on our [[BioMicroCenter:Tecan_Freedom_Evo|Tecan EVO150s]] and on our [[BioMicroCenter:Mosquito|TTP Mosquito.]] Libraries will be built to include variable i5 and i7 indexes based on the grid so dual indexing is required during sequencing of these libraries. For more information about how Nextera works, please check out the [[BioMicroCenter:DNA_LIB#NEXTERA_.2F_NEXTERAXT|standard Nextera library preparation section]] of [[BioMicroCenter:DNA_LIB|standard DNA library prep.]] | ||
Libraries prepared on the Mosquito use a significantly lower volume for preparation. This allows for significantly reduced costs but also lowers the complexity. As such, is it most suitable for single cell and amplicon analysis. De novo work should not be done using the reduced volume Mosquito preps. | |||
{|border=1 | {|border=1 style="text-align: center;" | ||
!colspan= | !colspan=4|Plate setup | ||
|- | |- | ||
| Batch Size || 16 | | Batch Size || 16 <BR> HT Standard NexteraXT|| 96 <br> Reduced volume XT|| 384 <br> Reduced volume XT | ||
|- | |- | ||
| Sample Layout || Columns from left | | Sample Layout || Columns from left || quads on 384w plate || full 384w plate | ||
|- | |- | ||
| Volume || >7ul | | Volume || >7ul || 5ul || 5ul | ||
|- | |- | ||
| Concentration || 0.2ng/ul* | | Concentration | ||
|colspan="3"| 0.2ng/ul* | |||
|- | |- | ||
| Buffer || H2O or 10mM Tris 8.0. <br> no organics! | | Buffer | ||
|colspan="3"| H2O or 10mM Tris 8.0. <br> no organics! | |||
|- | |- | ||
| Plate || Axygen 96well (CAT#) | | Plate || Axygen 96well (CAT#) | ||
|colspan="2"| Obtain plate from BMC | |||
|- | |- | ||
| Additional services available <BR> can be added if samples are not submitted as above | | Additional services available <BR> can be added if samples are not submitted as above | ||
| | |colspan="3"| | ||
* Sample setup (per 48) | * Sample setup (per 48) | ||
* SPRI cleanup (if not clean in proper buffer) | * SPRI cleanup (if not clean in proper buffer) | ||
* Advanced Analytical (if not quantified). | * Advanced Analytical (if not quantified). | ||
|- | |- | ||
|colspan= | |colspan="4"| * For short PCR fragments, <u>lower</u> concentrations are required. Please speak with BMC staff for amplicons. | ||
|} | |} | ||
<BR><BR> | <BR><BR> | ||
