Question

I just need the answers to questions 2 and 3. My DNA ladder is in lane 2 with the yellow arrow pointing to it. Thanks!

Part 2: Gel purification and ration Gel Slice and PCR Product Preparation modified from IBSci.com instructions for gel and PCR clean-up system A. Dissolving the Gel Slice 1. Following electrophoresis, excise DNA band from gel and place gel slice in a 1.5ml microcentrifuge tube. 1b. Use an analytical balance to weigh gel slice. Record weight here Md 500 DF togel Vortex and incubate at 50-65°C until gel slice is completely dissolved (10-15 min. then cool to room temp. Binding of DNA 1. Insert SV Minicolumn into Collection Tube 2. Transfer dissolved gel mixture to the Minicolumn assembly. Incubate at room temperature for 1 minute. 3. Centrifuge at 16,000 xg for 30 sec. Discard flowthrough and reinsert Minicolumn inte Collection Tube. Repeat with remaining Bel volume if needed Washing 4. Add 400 l W1 (ethanol added) Centrifuge at 16,000 xg for 30 sec. Discard flowthrough and reinsert Minicolumn into Collection Tube 5. Repeat Step 4 with 600 ul Wast Solution. Wait 1 minute. Centrifuge at 16,000 xg for 30 6. Empty the Collection Tube and recentrifuge the column assembly for 3 min. with the microcentrifuge lid open for off) to allow evaporation of any residual ethanol. Elution 7. Carefully transfer Minicolumn to a clean 1.5ml microcentrifuge tube. 8. Add 20ul of Nuclease-Free Water to the Minicolumn. Incubate at room temperature for 1 minute. Centrifuge at 16,000 xg for 1 minute. 9. Discard Minicolumn and store DNA at 4°C or -20°C. Additional protocol information is available in Technical Bulletin, available online at: IBISci.com Ligation Combine your gel-purified products following the recipe below. Your selection of compatible restriction enzyme sites should allow the parts to assemble in the correct order and orientation. 11 ul water 2 ul part 1 2 ul part 2 2 ul plasmid 2 ul ligation buffer 1 ul DNA ligase Total 20 ul Incubate 10 minutes to overnight at room temperature. Store at -20° C until transformation lab. Questions: 1. Sketch the results of your lane and the DNA ladder here, indicating the number of bands you can see and the approximate size (in base pairs) of your band(s), based on the DNA ladder. 30

Answer 1

2. After restriction if we get one band only then either the DNA is palsmid DNA that has been changed into linear form circular form or Restriction digestion has not taken place because enzyme was non-functional.

3. If restriction digests is mixed with DNA ligase but without pruifying the gel then no ligation will take place beacsue DNA liagse need DNA to be in its free form to perform ligation.