hi everyone, i will be attempting the first ever chip seq in our lab soon and i just have a bunch of questions that i can't find answers on and hoping someone with more experience could chime in.
this will be for yeast.
1. i've got my hands on a protocol that has me grow yeast in 50 ml to OD 0.7, then crosslink/formaldehyde and quench with glycine, per chip sample. does this seem about right?
2. for each pellet i collect from above, i actually want to chip with 2 different antibodies, so they'll be split in half. am i better of growing more cells or will that be enough DNA?
3. how can i ultimately test my 'efficiency'? or in other words, whether my chip worked? i know that after shearing, usually 1% is saved as an input. (and this is the step after which i plan on separating my 1 sample into 2 equal samples) then antibody is added to to pull down cross linked dna. after uncrosslinking and purification, can i just use qubit on the 1% and on the final purified stuff to see what my yield is like?
what is a typical yield in both ng and % of the input?
thanks a ton! i plan on using either miseq or hiseq with illumina.
this will be for yeast.
1. i've got my hands on a protocol that has me grow yeast in 50 ml to OD 0.7, then crosslink/formaldehyde and quench with glycine, per chip sample. does this seem about right?
2. for each pellet i collect from above, i actually want to chip with 2 different antibodies, so they'll be split in half. am i better of growing more cells or will that be enough DNA?
3. how can i ultimately test my 'efficiency'? or in other words, whether my chip worked? i know that after shearing, usually 1% is saved as an input. (and this is the step after which i plan on separating my 1 sample into 2 equal samples) then antibody is added to to pull down cross linked dna. after uncrosslinking and purification, can i just use qubit on the 1% and on the final purified stuff to see what my yield is like?
what is a typical yield in both ng and % of the input?
thanks a ton! i plan on using either miseq or hiseq with illumina.
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