DC3INSTAR
DNA is extracted by using phenol-chloroform-isoamyl alcohol. Quantity of extracted DNA is confirmed using Nanodrop 1000.Two readings were taken and further the average of these datapoints is noted. These results indicate that there is some amount of contamination of either proteins or chemicals like phenol etc. Therefore, to check the whether quality of DNA is sufficient to proceed further, electrophoresis is carried out. The quality of the quantified DNA was confirmed on the 1% Agarose gel. In brief,2 l of the DNA is mixed with 2 l of 2x loading dye (Invitrogen) and subjected to electrophoresis at 120 volts for 30 mins. DNA was purified by 3M sodium acetate followed by washing with 75% ethanol. Purified DNA quantity is confirmed using Nanodrop 1000, The quality of the PCR product was confirmed on the 2% agarose gel. V4-V5 primers were used for amplicon generation. In brief, 5 l of the PCR product was mixed with 1 l of 6x loading dye (Invitrogen) and subjected to electrophoresis at 120 volts for 30 minutes. Puregene Nexgen DNA ladder (PG210500 DI-NV) was loaded along with amplicons. Final libraries were quantified using Qubit 4.0 fluorometer (Thermofisher #Q33238) using DNA HS assay kit (Thermofisher #Q32851) following manufacturers protocol. Obtained results are presented below in the table. To identify the insert size of the library, we queried it on Tapestation 4150 (Agilent) utilizing highly sensitive D1000 screentapes (Agilent # 5067- 5582) following manufacturers protocol. Acquired sizes of all libraries are reported.