* PCR protocol Genotyping DNA in rat Dont forget to write down the protocol sheet before doing experiment,

prepare handschoen, micropippet tube, holder, and ice bag, for maintenance the reagent. 1. Mix @: 2xPCR buffer for KOD FX (10 microL), 2mM dNTPs (4microL), distilled water (add libitum). Primer 1(ku Com 0.6microL) primer 2ku KO(0.3microL), primer 3 (ku End 0.3microL), and last KOD FX enzyme(0.4microL) all ingredients in one tube. (Make sure all in soluble form, not iced) 2. Separated in to 5 tube (for example 4 become 5, in this experiment we using 4 tube) all including, except Genomic DNA 100ng. each tube contain 19microL of mix, back then adding 1 microL of genomic DNA. 3. Sentrifuge all 4 tubes, then writing down numbering and remind (example 92,93,93,95 for the specimen according left to right) with 75 speed. 4. Put in the PCR machine, turn on and make sure that step 3 Annealing must be 60 degree.(all step is denature 98 degree, annealing 60 degree, and extention 68 degree) set machine press (List_programs_PCR_ 3STE4KOD_ proceed_proceed_annealing 60 degree_remember change volume to 20 microliter_use heated lite)..wait for around 30-60 min 5. While you waiting make the agarose gel 6. Assembling the part of template, and then get TAE liquid 25ml using the automatic suction tube, and put in the bottle. And mix with 1.8% of agarose powder (1.8%x25ml=0.45gr) 7. Warming in microwave until soluble and soon pour in the square template with well. Before it getting hardened. Wait for 20-30 minutes. 8. After that unplug the the well template, and now the well will appear. Make sure that the electrophoresis place containing enough of TAE liquid. Other wise, the marker with not running. 9. First filling the ladder well in left side with 5 micro liter of ladder fluid GL 100(Gene Ladder 100) and then 10. Take and mix loading buffer 6x (1microL) with product PCR(5microL) 1:5 for tube number 92,93,94,95 in para film mix it using pipette, And then extract in the well one by one. Waiting for 20-30 minutes, till the bilayer appear, bilayer contains of bromophyl blue (dark blue)and etilen xylanol(light blue). 11. After that staining in etylen bromide, add 10 microL, becareful because it carcinogenic, waiting for 30 minutes 12. Look under the UV light, be careful to sweep mark of your hand, and then take pic with canon camera (turn on uv light, camera, and print )(press on_set_set_print) the interpretation will appearing the amount of base pair.

PCR Protocol genotyping DNA in Human 1. Write down the sheet and calculate the right dose. 2. In Human, we dont need destilled water. 3. And different temperature of annealing 58 derajat let the temperature for the extention time. 4. LIST= Program=main=XRCC-KOD, if you want to Check Protocol =RUN. 5. when make agarose gel using double, large box, with 50 ml TAE1x and 0.9 gr of powder 6. we make 20 microliter of DNA sample, and mix with 5 microliter of 6x loading buffer 20:5 7. and then make agarose gel with bromophyl blue/dark blue and etylen xylenol(light blue). 8.