TITLE: Genomic Assembly of Plasmid DNA from Bacterial Cultures
AUTHORS: Ryan Teague

[DESCRIPTION]
This protocol was developed for BIT 495: Special Topics in Biotechnology: Portable Genome Sequencing at North Carolina State University. 

            The purpose of this protocol is to consistently complete genomic assemblies of plasmid DNA from bacterial cultures. This is done by ensuring that the DNA is sequenced with long DNA reads, which helps the DNA preserve its integrity and decreases the chance of the sample having multiple contigs in the final assembly. This protocol has been optimized to produce high-quality reads from the most difficult-to-extract plasmid DNA. 

This protocol is broken down into a few key steps:
A bacterial colony is grown, and then the plasmids are extracted using the method given below
The DNA is purified from the plasmids by using a slight variation of the QIAprep Spin Miniprep kit 
The DNA library will be made using the Ligation Sequencing kit but with a custom protocol designed for plasmids
Load this sample into a MinION flow cell and use a MinION sequencer to extract the reads
The analysis of our samples will be completed using BV-BRC

Note: For the last two steps of this process (the sequencing and analysis) there will be no strict procedures given, only brief descriptions. 
 
 
 

[GUIDELINES]
When following this protocol, ensure that all steps are carried out under sterile conditions to avoid contamination of the plasmid DNA. Always use fresh reagents, and handle all materials with care to maintain the integrity of your samples. Pipette accurately and avoid excessive vortexing to prevent shearing of the DNA. Consistently monitor centrifugation speeds and times to ensure proper separation during plasmid extraction and purification. For DNA quantification, ensure calibration of equipment such as NanoDrop and Qubit Fluorometer. Follow manufacturer instructions when using commercial kits like the QIAprep Spin Miniprep kit and Ligation Sequencing Kit V14 to achieve optimal results. You may pause this process between any major section of this protocol. 

[STEPS]
SECTION: 1. Plasmid Extraction from Bacterial Colony
1. To start, obtain an overnight culture of bacteria (of whatever type of bacteria)
SECTION: 1. Plasmid Extraction from Bacterial Colony
2. Centrifuge the culture to pellet the bacteria
SECTION: 1. Plasmid Extraction from Bacterial Colony
3. Remove all supernatant and resuspend the bacteria in the resuspension buffer
SECTION: 1. Plasmid Extraction from Bacterial Colony
4. Add a denaturing solution to the resuspended bacteria 
This will lyse the bacteria to release their contents (including plasmid DNA)
SECTION: 1. Plasmid Extraction from Bacterial Colony
5. Add a renaturing solution to the denatured bacteria
This causes proteins and genomic DNA to precipitate while leaving smaller plasmids in the solution
SECTION: 1. Plasmid Extraction from Bacterial Colony
6. Pellet the proteins and genomic DNA by centrifugation, and remove the plasmid-containing supernatant
SECTION: 2. DNA Purification From Plasmid
7. Add an equal volume of absolute ethanol into each tube and mix by inverting the tubes a few times
SECTION: 2. DNA Purification From Plasmid
8. Centrifuge the tubes at 20,000× g (15,000 rpm) for 5 min
SECTION: 2. DNA Purification From Plasmid
9. Decant the supernatant and carefully remove the liquid completely using a pipette
SECTION: 2. DNA Purification From Plasmid
10. Add 300 μL of TE buffer (pH 8.0) (see recipe above), 150 μL of 5 M NaCl (see recipe above), and 45 μL of 20% SDS (see recipe above). Mix by inversion and incubate at 65 °C in a heating block for at least 10 min
SECTION: 2. DNA Purification From Plasmid
11. Add an equal volume (500 μL) of CIA (see recipe above) and keep shaking on a Nutating Mixer for at least 5 min
SECTION: 2. DNA Purification From Plasmid
12. Centrifuge at 20,000× g (15,000 rpm) for 7–10 min
SECTION: 2. DNA Purification From Plasmid
13. Carefully transfer the upper phase (~400 μL) into new 1.5 mL Eppendorf tubes, add 16 μL (20 mg/mL) of RNase, mix well by inversion, and incubate at 37 °C in a heated block for at least 15 min. Add an equal volume of CIA and keep shaking on a Nutating Mixer for at least 5 min
SECTION: 2. DNA Purification From Plasmid
14. Centrifuge at 20,000× g (15,000 rpm) for 7–10 min
SECTION: 2. DNA Purification From Plasmid
15. Carefully transfer the upper phase (300 μL) into new 1.5 mL Eppendorf tubes, add an equal volume of absolute ethanol, and mix well by inversion
SECTION: 2. DNA Purification From Plasmid
16. Centrifuge at 20,000× g (15,000 rpm) for 7–10 min
SECTION: 2. DNA Purification From Plasmid
17. Carefully decant or pipette out the liquid phase and add 500 μL of 70% ethanol. Tap the tube to release the pellet
SECTION: 2. DNA Purification From Plasmid
18. Centrifuge at 20,000× g (15,000 rpm) for 1–2 min and repeat step 11
SECTION: 2. DNA Purification From Plasmid
19. Add 50 μL (if the pellet is large, add more volume) of TE Buffer. Completely dissolve the pellet by finger-tapping
SECTION: 2. DNA Purification From Plasmid
20. Measure the quantity of the plasmid DNA using NanoDrop and using Qubit (this concentration will be helpful for the next steps
SECTION: 3.1 Library Preparation of DNA - DNA Repair and End Prep
21. Use the Qubit Fluorometer to find the concentration of plasmid DNA in your sample
SECTION: 3.1 Library Preparation of DNA - DNA Repair and End Prep
22. Adjust the volume/concentration to get 1 ug of plasmid DNA 
This ensures that long reads will be obtained in this process
SECTION: 3.1 Library Preparation of DNA - DNA Repair and End Prep
23. Transfer 1 μg input DNA into a 1.5 ml Eppendorf DNA LoBind tube
SECTION: 3.1 Library Preparation of DNA - DNA Repair and End Prep
24. Adjust the volume to 48 μl with nuclease-free water
SECTION: 3.1 Library Preparation of DNA - DNA Repair and End Prep
25. Mix thoroughly by pipetting up and down
SECTION: 3.1 Library Preparation of DNA - DNA Repair and End Prep
26. Spin Down with the microfuge 
SECTION: 3.1 Library Preparation of DNA - DNA Repair and End Prep
27. In a 0.2 ml thin-walled PCR tube, mix the following (pipette mix between each addition):
48 uL of the DNA sample 
2 uL of NEBNext FFPE DNA Repair Buffer v2
2 uL of NEBNext FFPE DNA Repair Mix
3 uL of Ultra II End-prep Enzyme Mix
SECTION: 3.1 Library Preparation of DNA - DNA Repair and End Prep
28. Mix the reaction and gently spin it down 
SECTION: 3.1 Library Preparation of DNA - DNA Repair and End Prep
29. Incubate with the Thermocycler at 20°C for 5 minutes and 65°C for 5 minutes
SECTION: 3.1 Library Preparation of DNA - DNA Repair and End Prep
30. Transfer the DNA sample to a clean 1.5 ml Eppendorf DNA LoBind tube
SECTION: 3.1 Library Preparation of DNA - DNA Repair and End Prep
31. Resuspend the AMPure XP Beads (AXP) by vortexing
SECTION: 3.1 Library Preparation of DNA - DNA Repair and End Prep
32. Add 60 µl of resuspended AMPure XP Beads (AXP) to the end-prep reaction and mix by flicking the tube
SECTION: 3.1 Library Preparation of DNA - DNA Repair and End Prep
33. Incubate on a Hula mixer for 10 minutes at room temperature
SECTION: 3.1 Library Preparation of DNA - DNA Repair and End Prep
34. Prepare 500 μl of fresh 80% ethanol in nuclease-free water
SECTION: 3.1 Library Preparation of DNA - DNA Repair and End Prep
35. Spin down the sample and pellet on a magnet until the supernatant is clear and colorless. Keep the tube on the magnet, and pipette off the supernatant
SECTION: 3.1 Library Preparation of DNA - DNA Repair and End Prep
36. Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard
SECTION: 3.1 Library Preparation of DNA - DNA Repair and End Prep
37. Repeat the previous step
SECTION: 3.1 Library Preparation of DNA - DNA Repair and End Prep
38. Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~15 seconds, but do not dry the pellet to the point of cracking
SECTION: 3.1 Library Preparation of DNA - DNA Repair and End Prep
39. Remove the tube from the magnetic rack and resuspend the pellet in 61 µl nuclease-free water. Incubate for 2 minutes at room temperature
SECTION: 3.1 Library Preparation of DNA - DNA Repair and End Prep
40. Pellet the beads on a magnet until the eluate is clear and colorless, for at least 1 minute
SECTION: 3.1 Library Preparation of DNA - DNA Repair and End Prep
41. Remove and retain 61 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube
SECTION: 3.1 Library Preparation of DNA - DNA Repair and End Prep
42. Quantity 1 uL of the eluted sample using a Qubit Fluorometer
SECTION: 3.2 Library Preparation of DNA - Adapter Ligation and Clean Up
43. Make note of the sample’s DNA concentration 
SECTION: 3.2 Library Preparation of DNA - Adapter Ligation and Clean Up
44. Thaw all reagents, individually spin them down, and store them on ice 
SECTION: 3.2 Library Preparation of DNA - Adapter Ligation and Clean Up
45. In a 1.5 ml Eppendorf DNA LoBind tube, mix in the following order:
60 uL of the DNA sample 
5 uL of the ligation adapter 
25 uL of the ligation buffer
10 uL of the Salt-T4® DNA Ligase
SECTION: 3.2 Library Preparation of DNA - Adapter Ligation and Clean Up
46. Thoroughly mix the reaction by gently pipetting and briefly spinning down
SECTION: 3.2 Library Preparation of DNA - Adapter Ligation and Clean Up
47. Incubate the reaction for 10 minutes at room temperature
SECTION: 3.2 Library Preparation of DNA - Adapter Ligation and Clean Up
48. Resuspend the AMPure XP Beads (AXP) by vortexing
SECTION: 3.2 Library Preparation of DNA - Adapter Ligation and Clean Up
49. Add 40 µl of resuspended AMPure XP Beads (AXP) to the reaction and mix by flicking the tube
SECTION: 3.2 Library Preparation of DNA - Adapter Ligation and Clean Up
50. Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature
SECTION: 3.2 Library Preparation of DNA - Adapter Ligation and Clean Up
51. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant when clear and colorless
SECTION: 3.2 Library Preparation of DNA - Adapter Ligation and Clean Up
52. Wash the beads by adding 250 μl Long Fragment Buffer (LFB). Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard
SECTION: 3.2 Library Preparation of DNA - Adapter Ligation and Clean Up
53. Repeat the previous step
SECTION: 3.2 Library Preparation of DNA - Adapter Ligation and Clean Up
54. Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow to dry for ~15 seconds, but do not dry the pellet to the point of cracking
SECTION: 3.2 Library Preparation of DNA - Adapter Ligation and Clean Up
55. Remove the tube from the magnetic rack and resuspend the pellet in a 15 µl Elution Buffer (EB). Spin down and incubate for 10 minutes at 37°C to improve the recovery of long fragments
SECTION: 3.2 Library Preparation of DNA - Adapter Ligation and Clean Up
56. Pellet the beads on a magnet until the eluate is clear and colorless, for at least 3 minutes
SECTION: 3.2 Library Preparation of DNA - Adapter Ligation and Clean Up
57. Remove and retain 15 µl of eluate containing the DNA library in a clean 1.5 ml Eppendorf DNA LoBind tube
SECTION: 3.2 Library Preparation of DNA - Adapter Ligation and Clean Up
58. Measure the concentration of the sample using the Qubit Fluorometer
SECTION: 3.2 Library Preparation of DNA - Adapter Ligation and Clean Up
59. Prepare your final library as at least 300 ng of Plasmid DNA into 12 uL of elution buffer 
This ensures long reads of the plasmid DNA 
SECTION: 3.2 Library Preparation of DNA - Adapter Ligation and Clean Up
60. The priming mixture is made as follows  in a 1.5 mL Eppendorf LoBind tube:
1170 uL of Flow Cell Flush  
5 uL of Bovine Serum Albumin (BSA) at 50 mg/ml
30 uL of Flow Cell Tether 
SECTION: 3.2 Library Preparation of DNA - Adapter Ligation and Clean Up
61. The Final Library mixture is made as follows in a 1.5 mL Eppendorf LoBind tube: 
37.5 uL of Sequencing Buffer 
25.5 uL of Library Beads (LIB) mixed immediately before use
12 uL of DNA library
SECTION: 4. Nanopore Sequencing
62. This section won’t be in detail, but the library sample will be inserted into the MinION flow cell, which will hook up to the GridION sequencer
Here is a video tutorial on priming and loading the MinION Flow Cell: https://www.youtube.com/watch?v=IknVaEnuDz0
SECTION: 5. Sequencing Analysis
63. This section will also not be in detail, but the results from the sequencing will be run into a comprehensive genome analysis on the BV-BRC website software