NOTE: There are concentration recommendations in this protocol. Don't ignore these. To do so WILL increase the chance of failure.

A printable version (PDF) of this protocol suitable for benchtop use is available at the top of this page.

Selecting DNA samples:
You will need a LARGE quantity (~12 µl) of genomic DNA for this procedure.
Get this from (I have used both successfully):

• -Extraction of a good piece of tissue (EtOH preserved works fine).
  
• -WGA your DNA using QIAGEN "Repli-G" or GE "GenomiPhi" kits.
  

Fragment DNA:
Digest 22 µl of genomic DNA (> 550 ng/µl) with a restriction enzyme (any 4 cutter should do but you should do a test digest to see that you get complete digestion. Example below is specific to RsaI. Modify as needed if you choose another.).

• Recipe (50 µl total volume)
  
  • 3 µl RsaI (10u/µl)
    
  • 25 µl 10X NEBuffer 1 (whichever buffer specified for your chosen enzyme)
    
  • 22 µl Genomic DNA
    
  
  
  
• -Incubate at 37C for 1 hour
  
• -Heat inactivate (temps vary, read enzyme documentation) enzyme (if storing) or run immediately on gel (see below).
  

Size Selection
As cloning will preferentially incorporate smaller fragments (that are useless for our purposes) you must run the entire extract out on a 1% agarose gel and cut out the 1-2 kb size range. Use a gel rig/thickness/comb combination that will allow you to load the entire 50 µl into a single (or at most 2) well.

After running this digest (with ladder!), cut out the portion of the smear that spans the 1-2 kb size range. NOTE: Be careful to limit the exposure of the gel to UV. This will nick the DNA and that is bad news.

• Use a QIAGEN (or whichever brand you like) Gel-Extraction kit to recover the fragmented DNA from your gel slice.
  • NOTES

    -Do not run the digest out too far. This will make your gel slice HUGE. -Your gel slice will likely be too large to dissolve in 500 µl of QG (do not use more than this). Cut the slice in half and dissolve in 2 separate 2.0 ml tubes. Run both of these through a single spin column (i.e. fill, spin, discard flow through, repeat until all of your QG+gel has gone through). -You can skip step 5 (Isopropanol) -DO the extra Buffer QG wash step. -Read Note of step 10. DO let the column stand after adding PE -Elute with 50 µl of H2O

    w

Cloning
Quantify the gel-extracted fragment concentration (I use a NanoDrop). For 1-2kb fragments you will need 5 µl at a concentration of 20 ng/µl or greater (> 100 ng of size selected DNA). This is the MINIMUM amt. as it gives a 10:1 molar ratio of insert:vector. Optimal efficiency occurs at 10:1 to 100:1. If your gel extracted, size selected DNA needs to be concentrated, simply put in a spin-vac (this is why we eluted with water above)

• The digested DNA fragments are blunt ended and are easily cloned with the Invitrogen Zero Blunt PCR Cloning Kit (Invitrogen Catalog # K270020) following the directions exactly.
  
  
• NOTES
• When transforming cells, be certain not to mix by pipetting.
• The length of the 42C heat shock (45 sec.) and the immediate transfer to ice are critical. No wiggle room here.
• Shaking of cells with SOC must be done in the shaking incubator at the recommended speed.
• Be sure that you use kanamycin as your selective agent.
• There is no color selection. All (80%+) colonies should have inserts.

Getting sequences
Theoretically, PCR with M13F (-20) and M13R primers should be possible with simply using a pipette tip to transfer cells from an individual colony to the PCR master mix. In reality, my success with this is very limited (~25%) picking colonies from the initial plates. I have had the best success replating colonies onto a grid and growing them up until they are good sized (2mm). A 15 µl PCR using the M13 primers and a wad of cells picked with a pipette tip works just fine. I typically get 75+ products from PCR of a 96 well plate. From these I sequence (in both directions) all fragments that fall in the 600-1500 bp range. This gives me plenty of room for primer development. I generally end up sequencing 45-60 from each plate of PCR.

Also, using a mini prep kit to isolate the plasmids works like a charm every time but is very $$$. I suggest trying the direct PCR method.

Amplify the insert (using either method mentioned above) and sequence using the M13 primers.





Identifying candidate loci:

• Sequences should first be compared to the NCBI database (using BLAST) to determine whether the locus is identifiable as a particular gene or locus (if so, toss it out).
• If sequences return no significant BLAST hit, use whatever you like to create primers. I use Geneious. PRIMER3 is a nice free option.
  
  
  • NOTES
  • -Despite size selection, it seems that small fragments sneak through.
  • -Dont bother sequencing inserts that are smaller than 500bp. These are not sufficiently large for subsequent development.

Getting Loci
Use these primers to screen a small number of individuals (usually 7 from all available portions of the range or previously identified haplogroups) for variation. Find it and you are good to go.