Question

I need the answers for questions 2 and 3. My DNA ladder is in lane 2 marked by the yellow arrow. Thanks!

Part 2: Gel purification and on Gel Slice and PCR Product Preparin modified from TBSci.com instructions for gaan A. Dissolving the Gel Stie Following electrophores, eral DNA band from grand place glice microcentrifuge tube Ib. Use an analytical balance to weigh pelice Rec die 2. Add 500 balance to weigh pelice Record w here 110-15 min, then cool to room temp 500 l Of togel Vortex and inte 50-69"Curvelice is completely dissolved Binding of DNA 1. Insert SV Minicolumn in Collection Tube 2. Transfer dissolved gelmisture to the Municum assembly incubate at room temperature for 1 minute 1. Centrifuge at 16,000 for 30 sec Discard towth and reset Minicolumn into Collection Tube. Repeat with remaininggel volume de Washing 4. Add 400 d Wiethanol added). Centrifuge at 16.000 for 30 sec Discard flow and risert Minicolumn into Collection Tube 16,000 for 30 5. Repeat Step with Gol West Solution. Wait minute. Centrifuge 6. Empty the Collection Tube and recentrifuge the column assembly for 3 min with the microcentrifuge lid open for of) to allow evaporation of any residual ethanol 7. Carefully transfer Minicolumn to a clean 15ml microcentrifuge tube 8. Add 20ul of Nuclease-Free Water to the Minicom. Incubate at room temperature for 1 minute. Centrifuge at 16,000 for 1 minute 9. Discard Minicolumn and store DNA at 4'Cor -20°C. Additional protocol information is available in Technical Bulletin, available online at: Sci.com STTTTTTTTEE 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 l part 1 2 u part 2 2 ul plasmid 2 ul ligation buffer Il DNA ligase Total 20 l 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

Here is the only other info I have. Thanks!

Answer 1

2. a. One possibility can be that, one of the enzymes did not work properly, that is did not cut the DNA, and thus, only one cut resulting from one of the enzymes gave rise to only one linear form of the DNA, giving one band.

  b. Another possibility is that, (if both the fragments are not of the same molecular weight) the smaller of the two fragments was so small that it ran out from the gel.

3. Gel-purification is essential before ligation, as it is important to remove all the agarose, and all other buffers, chemicals present in the DNA solution, to make sure that the ligase works properly in its own buffer. If purification is not done, then there will be faulty ligation, as the enzyme will fail to work properly due to the interference from the other components present already. There might also be some self-ligated products seen.