Skip to content

gel_extraction_protocol.md

Latest commit

 

History

History
69 lines (64 loc) · 6.82 KB

gel_extraction_protocol.md

File metadata and controls

69 lines (64 loc) · 6.82 KB
Raw

Gel Extraction Protocol

This protocol is used to select fragment sizes for pooled PCR products produced from our multiplexed DNA library preparation protocol. For conifers, we generally cut a bandwidth covering between 300-500bp.

Prep 1:

  • Before you start make sure there is enough 1x TAE buffer. You will need about 1000ml, if there isn’t enough make a fresh stock using the 10x TAE buffer and Autoclaved water.
  • To make 1x TAE follow the "CASTING GEL" protocol on github and to make autoclave water follow "AUTOCLAVE WATER" protocol
  • Once you have made sure there is enough 1x TAE buffer follow through the "CASTING GEL" protocol and prepare the 1% agarose gel.

Prep 2:

  • Take the 100bp DNA ladder and the 6X blue dye out from the -20 freezer and let it thaw. Once thawed place on ice.
  • Label a blue-capped 15mL centrifuge tube as Pooled PCR product of [sample X] [date] [initials].
  • Centrifuge the two PCR product plates (they should be in the fridge) to let the products settle at the bottom.
  • Combine all of the wells from the two PCR product plates into the 15mL tube. To do this use a multichannel pipette (set at 20ul) and transfer the PCR products to a reservoir. Now use a 1000ul pipette and transfer everthing to the 15ml tube.

Main Protocol adapted from QIAquick® Gel Extraction Kit:

  1. Place 1% agarose gel in electrophoresis apparatus and make sure that it is completely submerged in 1x TAE buffer.
  2. On a piece of wax paper or in a 96 well plate used for PCR place two 3μL drops of loading dye.
  3. After dye has been placed, add 3μL of DNA ladder to each of the two drops of dye. Mix the dye and the ladder by pipetting up and down gently a few times.
  4. Add the first dye/ladder drop to the first lane of the gel. MAKE SURE THERE IS NO BUBBLE IN THE TIP.
  5. Add the second dye/ladder drop to the 14th lane of the gel
  6. Into a 1.5mL centrifuge tube combine 710μL of pooled PCR product and 36μL of loading dye
  7. Add 60μL of the dye/PCR-product mix to each of lanes 2-13
  8. Run gel for 1 hour at 110V
  9. After running gel, label 12 microcentrifuge tubes 1-12. At this time also turn on the water bath and set it to 50*C (setting = 5)
  10. Using a scalpel or razor blade, cut out the section of the gel between 300-500bp while over UV light. You will have to take all the required equipments to cut the gel to the Dyer lab on 2nd floor. While you are outside the lab make sure you don't touch anything with gloves on. TAKE YOUR GLOVES OFF.
  11. Make sure to minimize exposure of DNA to UV light while slicing the wells.
  12. Cut out each of the 12 lanes from the excised gel section and remove from UV light
  13. One at a time, place each excised lane onto UV light and trim off excess gel (above, below, and to the sides of fluorescence) from each, minimizing exposure of DNA to UV light
  14. Place each lane into one of the 12 labeled microcentrifuge tubes; order does not matter. Now take these back to the eckertlab and weigh the gel slices
  15. Zero the scale with an empty 1.5mL microcentrifuge tube
  16. Place each of the 12 microcentrifuge tubes containing excised lanes on the scale and record mass in lab notebook with respective label of tube
  17. Generally, each trimmed lane is between 0.1-0.2g (this will help maximize use of extraction kit)
  18. Add 3 volumes of QG buffer to each tube and incubate at 50*C in waterbath until completely dissolved.
  19. For example, for a 100mg (0.1g) sample, add 300μL of QG buffer
  20. Vortex each sample every couple of minutes to expedite the dissolution
  21. Once dissolved, ensure that the color of the mixture is yellow (should be same color as QG buffer). If the color of the mixture is orange or violet, add 10μL 3M NaAc and mix. This should return to yellow color.
  22. Spin each microcentrifuge tube in the microcentrifuge for 5s to remove sample from lids
  23. Pour 5 mL isopropanol into a beaker
  24. Add 1 volume isopropanol to each microcentrifuge tube containing the dissolved lanes
  25. For example, for a 100mg (0.1g) sample, add 100μL of isopropanol
  26. Calculate how many of the purple spin columns will be necessary
  27. The purple spin columns are the limiting factor for these kits (i.e., the number of samples that can be processed from this kit is limited to the number of included spin columns - there has always been sufficient excess of buffers etc)
  28. Assign excized lanes to spin columns
  29. Each spin column can hold up to 400mg of gel. Combining multiple gel-excised lanes into a single spin column is ok as long as the total gel mass between the lanes does not exceed 400mg. Make sure to note in a notebook which lanes will be added to which spin column; make sure to label each spin column.
  30. Assign excised lanes across spin columns as to minimize number of spin columns used; usually 6 spin columns will be sufficient
  31. Add (at most) 750μL of (any of) the assigned QG-buffer/excised-gel solution to an assigned spin column. A larger volume will splash when closing the spin column lid.
  32. Centrifuge at 17900xG for 1 minute
  33. Discard the flow through
  34. If more of the QG-buffer/excised-gel solution remains, repeat until no samples are left. Make sure to spin down the QG-buffer/excised-gel solution tubes to ensure that all of the samples have been put through the spin columns
  35. Add 500μL of buffer QG to each spin column and centrifuge at 17900xG for 1 minute, discard flow through
  36. Add 750μL of buffer PE(Ensure addition of EtOH) to each spin column and let stand for 5 minutes
  37. Centrifuge columns at 17900xG for 1 minute, discard flow through
  38. Centrifuge spin columns once more to remove any residual buffer, discard flow through
  39. Place each spin column into a clean 1.5mL microcentrifuge tube; unnecessary to label the tubes
  40. Add 25μL of buffer EB to the white center of the spin column membrane – this step will elute the DNA, if buffer EB is on the side of the spin column it cannot perform at max potential.
  41. Let stand for 4 minutes
  42. Centrifuge at 17900xG for 1 minute DO NOT DISCARD FLOW THROUGH!
  43. Add another 25μL of buffer EB as in step 19
  44. Let stand for 4 minutes
  45. Centrifuge at 17900xG for 1 minute DO NOT DISCARD FLOW THROUGH!
  46. Combine all elution samples into a single 1.5mL microcentrifuge tube
  47. Label as “Gel-extracted [sample X] [date] [initials] [total volume of elution]"
  48. It is absolutely necessary that the total volume is known, as this will be needed if the DNA Concentration Protocol is required.
  49. Nanodrop Sample
  50. If total DNA concentration < 10 ng/μL, use speedVac concentration protocol and tell Dr. Eckert or whoever is incharge at that time