Difference: ProceduresKappaNgsPrep (31 vs. 32)

Revision 322018-09-12 - GabrielSuarez

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Major version notes

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  1. Low retention (maximum recovery) tubes and filter tips should be used at every step. Regular tips commonly used for EtOH removal during wash steps.
  2. Before starting make sure ligated adapters are available in cardboard box labeled 'Anealed adapters'.
    ALERT! Importance of this box/these tubes never being warmed above RT cannot be underscored enough
  3. WFI water should be used for all steps where water is needed. Aliquots of water should be taken from bottle to avoid contamination.
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  1. A 2 barcode system comprising internal and external barcodes is currently being employed (it allows to mix different samples in a single tube for multiplexing the sequencing, thus economical; it is different than dual barcoding strategies which use 2 different external barcodes). There are 8 annealed adapters, each corresponding to 1 of 8 internal barcodes (IBC). External barcode(s) (EBC) are added to samples in the PCR step. Identical EBC can and should be pooled together to meet the sequencing core's minimum requirement of 10million reads per sample this should give ~70-80x coverage of E. coli genomes which is the current standard.
    ALERT! NEVER pool identical IBC as this type of pooling can not be reassigned at the data analysis step.
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  1. A 2 barcode system comprising internal and external barcodes is currently being employed (it allows to mix different samples in a single tube for multiplexing the sequencing, thus economical; it is different than dual barcoding strategies which use 2 different external barcodes). There are 8 annealed adapters, each corresponding to 1 of 8 internal barcodes (IBC). External barcode(s) (EBC) are added to samples in the PCR step. Identical EBC can and should be pooled together to meet the sequencing core's minimum requirement of 10million reads per sample this should give ~70-80x coverage ("single-end" reads; see Coverage calculator) of E. coli genomes which is the current standard.
    ALERT! NEVER pool identical IBC as this type of pooling can not be reassigned at the data analysis step.
 
  1. Sample should never be vortexed, always thoroughly mixed by pipetting. This is done to avoid sample getting near the top of the tube and increasing contamination risk during magnetic bead clean up.
  2. "Freshly prepared 80% EtOH" for cleanup reactions should be prepared daily, and be made combining 100% EtOH with WFI water.
  3. Always have the next master mix prepared to add to the tubes after wash. Never leave the beads in tubes without liquid on them for very long (< 3 min) as they will dry and ruin your prep.
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  1. Set heat block (with water on the wells) to 37°C and corroborate temperature with a thermometer. *Beware some heat-blocks may have a timer and will stop prematurely if not set properly.
  2. Combine the following in a PCR tube:

Item Amount
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DNA 1 µg
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DNA 500 ng (minimal) - 1 µg (ideal)
 
Fragmentase 10x buffer 2 µl
Fragmentase enzyme mix (last ingredient to add) 2 µl
diH2O complete to 20µL
 
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