I want to sequence various strains of Bacillus ssp with the PacBio Multiplex Sequencing Protocol.
As you need 1 ug of DNA per strain, we use RotiPinkDNA (linear polyacrylamide with a dye) and glycogen toimprove DNA recovery of the Lucigen Total DNA/RNA extaction kit.
DNA Extraction Protocol:
1 mL of overnight BI-broth were resuspended in 150 µL TE buffer containing 1250 U of Ready-Lyse Lysozyme Solution (Lucigen, Middleton, WI, USA) and then incubated at 37 ◦C for 60 min. Subsequently, 150 µL of Tissue and Cell Lysis Solution containing 1 µL of Proteinase K (50 µg/µL) were added. The mixture was incubated for 15 min with shaking at 900 rpm at 65 ◦C, and vortexed every 5 min. After placing the samples on ice for 5 min, 175 µL of MPC Protein Precipitation Reagent were added. Cell debris was pelleted by centrifugation at 4 ◦C for 10 min at 13,100× g in a microcentrifuge. The supernatant was transferred to a fresh microcentrifuge tube and the pellet was discarded. A volume of 5 µL Roti-Pink (Carl Roth, Karlsruhe, Germany), 10 µL glycogen solution (5 mg/mL, Carl Roth, Karlsruhe, Germany), and 500 µL isopropanol (Carl Roth, Karlsruhe, Germany) were added to the recovered supernatant and then carefully mixed by inverting the tube several times. The precipitated DNA was pelleted by centrifugation at 4 ◦C for 10 min at 13,100× g in a microcentrifuge. After washing DNA pellets twice with 200 µL of 70% ethanol, the DNA was resuspended in 50 µL TE (10 mM Tris-HCl [pH 7.5], 1 mM EDTA) buffer and then stored at −20 ◦C until further use. (Basically this Publication Article Evaluation of a Highly Efficient DNA Extraction Method for B...
DNA Shearing:
1 ug of DNA is suspended in 100 uL of PacBio Elution Buffer and subjected to DNA shearing with the MegaRuptor 2.
Then, the protocol follows the Microbial Multiplexing Protocol from PacBio.
For the final prepped libraries (without polymerase) I have rather unpure libraries:
260/280 260/230
Cell3 1,69 1,11
Cell 4 1,83 1,58
How can I improve this libraries? If possible at all?
In the end, I get 87,7% P0, 3,1% P1 and 9,2% P2 (Cell 1) as well as 94,6 P0, 2,3% P1 and 3,1% P2 (Cell 2). Which really does not make sense to me at all.
So to my question:
Is it more likely, that RotiPinkDNA/Glycogen screwed up the sequencing, did we screw up the library prep or is it something other?
Best regards
- Badges
- Science method