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can someone answer my doubts because I would like to improve my protocol:
i shear 5ug of genomic dna on the covaris to 400bp. i get a good fragment size distribution in the 400bp-500bp range, where most of the material is concentrated in this range. there is some gDNA in the higher MW range and low MW range. however, i hope that the adapter-ligation sizeselection step later on eliminates that. using qiaquick after this fragmentation step cleans up some of this low MW dna.
next, i perform end repair, purify with qiaquick column and run some eluted end-repaired dna on a gel. there looks to be plenty here. next, i do 3'dA extension, purify with minelute column, and run some eluted 3'dA extended material on a gel.
questions here are:
-though it may not be discernable on a gel, there will be a shift in bp on the
3'dA gel compared to the end-repaired gel correct, due to the one base addition of A? so the 3' dA gel has a slight shift upwards? does anyone know exactly by how much?
next, i perform adapter ligation and purify with qiaquick column. i run some of the purified material on a gel for sizeselection. i see most of the material is concentrated in the 400-500bp region, so this is good. i do see some higher and lower MW material.
questions here are:
the lower MW material, if below 200bp is what i can safely assume is adapter dimers, single adapters that didn't ligate, ssDNA, or unligated unpolished dna that did not correctly get cleaned up in earlier steps correct? is that all? and what is the higher MW material? it could be unligated unpolished dna that did not get cleaned up, but could it be anything else? would size-selecting after fragmentation reduce this issue, or would the final yield of library reduce if size-selection after fragmention was first performed (instead of at the adapter-ligation step)?
another question is about chimeric fragments. i've read sizeselection after fragmentation and also after adapter ligation reduce chimeric fragments, which is two sizeselections in one prep, so how good would the final library yield be? has anyone optimized their genomic library prep protocol to reduce chimeric fragments, and how so?
fianlly i sizeselect the 400-500bp by cutting out a gel slice, purify the gel slice, and do 18 cycle PCR. i see a band at the 400-500bp range, so the PCR worked.
can anyone address these questions? they would be of great help to me. please don't just refer me to the literature papers such as those from Quail et. al in Nature Methods. I've read them, and none address the questions I am asking here.
i shear 5ug of genomic dna on the covaris to 400bp. i get a good fragment size distribution in the 400bp-500bp range, where most of the material is concentrated in this range. there is some gDNA in the higher MW range and low MW range. however, i hope that the adapter-ligation sizeselection step later on eliminates that. using qiaquick after this fragmentation step cleans up some of this low MW dna.
next, i perform end repair, purify with qiaquick column and run some eluted end-repaired dna on a gel. there looks to be plenty here. next, i do 3'dA extension, purify with minelute column, and run some eluted 3'dA extended material on a gel.
questions here are:
-though it may not be discernable on a gel, there will be a shift in bp on the
3'dA gel compared to the end-repaired gel correct, due to the one base addition of A? so the 3' dA gel has a slight shift upwards? does anyone know exactly by how much?
next, i perform adapter ligation and purify with qiaquick column. i run some of the purified material on a gel for sizeselection. i see most of the material is concentrated in the 400-500bp region, so this is good. i do see some higher and lower MW material.
questions here are:
the lower MW material, if below 200bp is what i can safely assume is adapter dimers, single adapters that didn't ligate, ssDNA, or unligated unpolished dna that did not correctly get cleaned up in earlier steps correct? is that all? and what is the higher MW material? it could be unligated unpolished dna that did not get cleaned up, but could it be anything else? would size-selecting after fragmentation reduce this issue, or would the final yield of library reduce if size-selection after fragmention was first performed (instead of at the adapter-ligation step)?
another question is about chimeric fragments. i've read sizeselection after fragmentation and also after adapter ligation reduce chimeric fragments, which is two sizeselections in one prep, so how good would the final library yield be? has anyone optimized their genomic library prep protocol to reduce chimeric fragments, and how so?
fianlly i sizeselect the 400-500bp by cutting out a gel slice, purify the gel slice, and do 18 cycle PCR. i see a band at the 400-500bp range, so the PCR worked.
can anyone address these questions? they would be of great help to me. please don't just refer me to the literature papers such as those from Quail et. al in Nature Methods. I've read them, and none address the questions I am asking here.
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