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DNA EXTRACTIONS 1. In an organic extraction, what would happen if you forgot to put SDS into the lysis buffer? 2. With the FTA punch-and-wash method, the DNA remains on the punch, even during subsequent reactions, such as amplification. Do you think that one punch (washed) can be used for both a real— time PCR quantitation & human identification locus amplification? Why or why not? 3. When using the Chelex method, how would accidentally pipetting some of the beads into an amplification reaction affect the results? 4. Silica column-based DNA extractions are very popular in forensic labs. These silica column methods are based on the affinity of the DNA to bind to silica under certain conditions. a. What are those conditions? b. Typically, the “elution buffer” (provided by the manufacturer) is used for final elution of the DNA off of the column. Could you elute your DNA off of the silica column using tap water instead of the commercial elution buffer? Why or why not? 5. When analysts perform a differential extraction for isolation of sperm cells in a sexual assault sample, typically the analyst will wash the sperm pellet after removing the “female” or nonsperm fraction but before completing the sperm cell lysis step. Why is this helpful? What would happen if you chose not to wash your sperm pellet? How would that affect your sperm and nonsperm fractions? 2 AMPLIFICATION 6. PCR a. Primer Design: Take the DNA sequence below, and choose a sequence to amplify whose product is 150bp. Define that sequence in brackets. Define the Forward & Reverse Primers, and write the sequence for each primer. 1 ggatcctgga accagagtta cagaaaggtg tgagctgctg tgtgggtgct aggaatcaaa 61 cctgggtctt ttagaggagc agccagtgca tttagctgct gagccatctc tcgggccctt 121 agttcaagtg ttttaaatga tgcattttat gctttgtata cgtaatgaaa taaaatgatt 181 tttttcccag aaaagattaa ggagcccact aaagactatg taactagacc gtggccaagc 241 cagatttgaa cagaacctgc aatgtctctt gaatggaaac tatgcatacc tgagcagcca 301 ccaaaattta ggacatccgg ttcttggcat ccctgtcttg tgtgacctta cacagactga 361 cctttgaaga tctcgttctc ttcagctatt aaataataag acttaagtat ctttgacggg Forward (5’ – 3’): Reverse (5’ – 3’): a. Describe your reasoning for selecting these primers over other potential sequences. b. During amplification, what would happen if you forgot to put in the MgCl2 intothe amplification reaction? 3 DNA QUANTITATION 7. Real-Time PCR (qPCR) a. Based on the amplification plot given below: i. What conclusions can you make regarding Sample A? ii. Which sample has a higher starting DNA concentration, Sample B or C? How do you know this? 4 b. Name one hallmark of a “good” standard curve, and explain why is it important to have this for a good standard curve? c. Based on the data given below: i. Estimate the concentrations of samples B & C: Sample Ct 100 ng/ul std 15 50 ng/ul std 16 25 ng/ul std 17 12.5 ng/ul std 18 6.25 ng/ul std 19 Sample A 19 Sample B 16.5 Sample C 15.25 5 8. UV-Spectrophotometry a. Calculate Concentration, total yield, and the OD260/OD280 ratio on the following sample: Qiagen DNA Extract eluted into 75ul. Dilution was made as follows: 20ul in 380ul ddH20 Data: OD260: .0050 OD280: .0029 9. (Slot-Blot STANDARDS (10ng to 0 ng, descending) a. What is your interpretation of this Quantiblot (slot blot) result? Did it work? Are the standards visible? Is the data usable? Why or why not? b. How could you improve this data? 6 10. Typically, 5ul of DNA extract is required for loading onto a slot-blot based quantitation assay. Based on the standards shown below, estimate quantity shown and concentration for the following samples: 11. Yield Gel Electrophoresis a. Lanes 1 & 13 are molecular weight ladders. Lanes 2 – 8 are DNA standards (250, 125, 62.5, 31.2, 15.6, 7.8, and 3.9 ng respectively). How do you interpret the data shown for samples in lanes 9, 10, 11, and 12? B) A: B: 7 b. You loaded 5 ul of each sample into a 1% agarose gel, and ran it at 75 volts for 45 minutes. Lane 1 is a ladder. Lanes 2 – 5 are the DNA standards (125, 100, 75, 50 ng from left to right). Can you determine quantity for lane 6? If so, what is the quantity of DNA in well 6? What is the estimated concentration of the sample from well 6? 12. See yield gel below. Given that there are no DNA standards included on this gel, can concentration for these samples be calculated? Can you make any conclusions regarding the quality of the samples? (LH and LD are molecular weight ladders).

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1. In an organic extraction, what would happen if you forgot to put SDS into the lysis buffer?
Answer: Sodium dodecyl sulfate (SDS) is an anionic detergent that helps in chemical lysis of cell membranes. It totally denatures protein including DNAse and RNAse enzymes as well as membrane proteins. SDS helps in emulsification of membrane lipids and disrupts the polar interactions with proteins. It forms complexes with proteins and lipids that cause precipitation of these materials out of the solution. So, if SDS is not added into lysis buffer, yield of extracted DNA will be low and quality will be poor too due to presence of nucleases.

2. With the FTA punch-and-wash method, the DNA remains on the punch, even during subsequent reactions, such as amplification. Do you think that one punch (washed) can be used for both a real— time PCR quantitation & human identification locus amplification? Why or why not?

Answer: The same punch cannot be used for both real-time PCR quantitation & human identification locus amplification.
After real-time PCR, huge amount of amplified dsDNA will remain attached on the punch that will interfere the binding of probe during human identification locus amplification....
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