Inter simple sequence repeats (ISSR) uses microsatellite sequences as primers in PCR to generate multilocus markers that vary between organisms. Analysis and comparison of ISSR’s between organisms can reveal relatedness

Protocol:

Used ISSR markers (OMAR – (GAG)^4 RC) and psbA

On Samples (Left->Right)

GWC01 , CRA05 , PRO01 , PFR01 ,PRM01

20 tube Master mix for ISS (1 microliter template per tube)

ddH20 -> 250 microliters

10x buffer + Mg -> 60 microliters

BSA -> 20 microliters

dNTPs ->40 microliters

OMAR Primer -> 5 microliters

Taq -> 5 microliters

20 tube Master mix for psbA with following volumes in each tube

ddH20 -> 15.4 microliters

10x buffer + Mg -> 2 microliters

BSA -> 1 microliters

dNTPs ->.20 microliters

F Primer -> .2 microliters

R Primer -> .2 microliters

Taq -> .04 microliters

Placed on thermocycler

Protocol:

Used extracted DNA from the first Plant DNA extraction lab

GWC01-GWC05

1. Loaded agarose gels with 3 microliters of template and 1 microliter of loading dye on parafilm
2. ran for about 25 minutes at 130 v in the first bottom row of gel 2

Preparing template DNA:

1. used two .2 ml pcr tubes
2. pipetted 1 microliter of each sample into respective tube
3. created master mix of (per tube) using primers of pSBA and ITS genes
   1. ddh20 – 15 microliters
   2. 10x buffer+mg – 2 μL
   3. BSA – 1 μL
   4. dNTPs – .20 μL
   5. f primer – .20 μL
   6. r primer – .20 μL
   7. Taq – .04 μL
   8. template – 1 μL
4. Pipetted master mix into respective tubes and ran on thermocycler

Used Geneious and jmodeltest to analyze sequenced DNA

Best Model based on AIC -> TPMuf+G

Best Model based on BIC -> HKY+G

Using Mr. Bayes my tree looked like this:

Processed

GWC01

GWC02

GWC03

GWC04

GWC05

From Grey Whale Beach Site 3

Protocol:

1. Labeled 1.5 ml centrifuge tubes with correct sample
2. Added 3 sterile 3.2-mm stainless steel beads to each tube
3. Added small amount of leaf tissue to each tube, wiped between
4. Loaded tubes within a tube rack into the modified reciprocating saw rack and mounted rack into saw
5. spun tubes down about 20 seconds on fast speed
6. added 434 microliters of preheated grind buffer to each tube
7. incubate buffered grandame at 65 degrees celsius for 10 min in water bath, mixing tubes by inversion every 3 mins
8. add 130 microliters of 3M pH 4.7 potassium acetate, invert tubes several times and incubate on ice for 5 min
9. Spin in a centrifuge at maximum force (about 14000-15000 rams) for 20 min
10. Label new 1.5 ml tubes with sample ID
11. Add 1.5 volumes binding buffer
12. Add 650 microliters of mixture from step 11 to epoch spin column tubes and centrifuge for 10 min at 15,000 rpm in a centrifuge and discard flow-through
13. repeat step 12 with the remaining volume from step 11
14. wash with 500 ml of 70% EtOH then centrifuge at 15000 rpm until all liquid has passed through. discard flow-through
15. repeat 14
16. centrifuge columns at 15000 rpm for 5 more mins
17. discard tube then place columns in 1.5 ml labeled micro centrifuge tube
18. add 100 microliters preheated sterile h20 to each tube. let stand for 5 mins then centrifuge for 2 min at 15000 rpm to elute DNA

Lab Exercises

1. 1. JPCC – Supposedly Black Snapper (Macolor niger); actually Gilt-Head Bream (Sparus aurata)
   2. JPCD (Only fwd, rev didn’t work) – Supposedly Albacore Tuna (Thunnus alalunga); actually Southern Bluefin Tuna (Thunnus maccoyii)
   3. JRP3 – Yellowfin Tuna (Thunnus albacares)
2. No polymorphisms in samples that worked