I made a plant organelle DNA library of 5 samples using the Nextera XT kit. A bioanalyser run prior to Normalization shows fragments 200 bp onwards (less than 200 also in some samples) with a peak around 1500 bp( attachment bioanal 1). Since I'll be using a Miseq v2 reagent kit(300cycles PE), I need to size select.
Q1. Is the size range of 200-500bp OK ?
Q2. How will fragments of <200 bp affect cluster generation?
Q3 . What protocol shall I use for size selection?
a)I tried double SPRI (1x followed by 0.55X + 0.12X), and though it was effective in removing <200 bp I got nothing in the final eluent after 0.12X.
b)I also tried eluting with E gel size select agarose gel(Invitrogen) in MQ water and collected fractions of 288,363,450,563bp ( attachment bioanal 2- Samples 1,2,3 have been eluted from sample 3 of bioanal 1)
Q4. Can these be used further? Nowhere have I come across e-gel being used -I wonder why?
Q5. If these can be used, then
a) fraction of what size(s) can be taken/ pooled?(Each fraction is eluted in 25ul)
b) What should be the consideration for the minimum concentration (for eg.the 288bp fragment is 1403pmol/l in sample 1) that can be used for proceeding with the normalization which is the next step. Since normalization of concentartion is done using the beads provided in the kit by Illumina, I don't know how this will work across samples.Particularly since I need to load 20 indexed libraries in one flow cell.
Thanks
Q1. Is the size range of 200-500bp OK ?
Q2. How will fragments of <200 bp affect cluster generation?
Q3 . What protocol shall I use for size selection?
a)I tried double SPRI (1x followed by 0.55X + 0.12X), and though it was effective in removing <200 bp I got nothing in the final eluent after 0.12X.
b)I also tried eluting with E gel size select agarose gel(Invitrogen) in MQ water and collected fractions of 288,363,450,563bp ( attachment bioanal 2- Samples 1,2,3 have been eluted from sample 3 of bioanal 1)
Q4. Can these be used further? Nowhere have I come across e-gel being used -I wonder why?
Q5. If these can be used, then
a) fraction of what size(s) can be taken/ pooled?(Each fraction is eluted in 25ul)
b) What should be the consideration for the minimum concentration (for eg.the 288bp fragment is 1403pmol/l in sample 1) that can be used for proceeding with the normalization which is the next step. Since normalization of concentartion is done using the beads provided in the kit by Illumina, I don't know how this will work across samples.Particularly since I need to load 20 indexed libraries in one flow cell.
Thanks
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