Primers 2 primers are required for exponential amplification Forward primer Primer whose sequence is the same as that of the template indicated Reverse primer Primer whose sequence is complementary to that of the template indicated Example 3’-GCGGTT••••••••••ATCGTTA-5’ Forward primer: ATTGCTA Reverse primer: CGCCAA

Mass and Molar Ratios of DNA You have a 5 Kbp recombinant plasmid which represents a 2 Kbp insert cloned in a 3 Kbp vector. How many µg of plasmid would you need to digest to obtain 0.5 µg of the insert? Ratio of plasmid/insert = 5/2 = 2.5 Need 2.5 X 0.5 µg = 1.25 µg plasmid to obtain 0.5 µg of insert

Mass and Molar Ratios of DNA You have a 12 Kbp recombinant plasmid which represents a 10 Kbp insert cloned in a 2 Kbp vector. How many µg of plasmid would you need to digest to obtain 0.5 µg of the insert? Ratio of plasmid/insert = 12/10 = 1.2 Need 1.2 X 0.5 µg = 0.6 µg plasmid to obtain 0.5 µg of insert

Mass and Molar Ratios of DNA You wish to setup a ligation between a 5 Kbp insert and 50ng of a 2.5 Kbp vector. What mass of insert must be added to the ligation mixture to have a molar ratio of insert:vector of 3:1? Mass ratio: vector/insert = 2.5/5 = 0.5 50ng insert : 50ng vector = Molar ratio of 0.5:1 50ngX insert : 50ng vector = Molar ratio of 0.5X:1 = 3:1 0.5X = 3; 3/0.5 = 6 6 X 50ng =300ng

Mass and Molar Ratios of DNA You wish to setup a ligation between a 0.5 Kbp insert and 50ng of a 2.5 Kbp vector. What mass of insert must be added to the ligation mixture to have a ratio of insert:vector of 3:1? Mass ratio: vector/insert = 2.5/0.5 = 5.0 50ng insert : 50ng vector = Molar ratio of 5.0:1 50ngX insert : 50ng vector = Molar ratio of 5.0X:1 = 3:1 5.0X = 3; 3/5.0 = 0.6 0.6 X 50ng =30ng

Midterm February 12-16th You will need to register for a time slot 1-3:30 PM 3:45-6:15 PM No electronic devices will be allowed Laptops, tablets, cell phones, etc. Lab computers and access to the internet is allowed Any form of communication is forbidden Network activity will be monitored

Midterm Breakdown Part I – (16 points) Part II – (6 points) 8 calculations Time estimate: 30 minutes Part II – (6 points) 6 bioinformatics exercises Part III – (5 points) 5 theory questions Time estimate 20 minutes Multiple choice Fill in the blank True or false

Midterm Breakdown Part IV – (10 points) 2 out of 3 problems Time estimate: 45 minutes Problem 1: 5 questions Gel analysis Restriction digests Restriction mapping Problem 2: 5 questions PCR & Cloning 8

Midterm Breakdown Problem 3: 5 questions Restriction mapping Southern analysis

Need to know DNA concentration constant Converting units Forward vs reverse primers Palindromes Bioinfo theory pUC vectors

Sample Questions You have a DNA solution with an absorbance at 260nm of 0.15. 0.4mL of this DNA solution is added to 0.6mL of water. What is the new concentration of DNA in mg/mL? The concentration of a salt solution is 0.38% (m/v). 124 mL of this solution is placed in a beaker and allowed to evaporate until 92 mL remains. What is the new salt concentration in mg/ml? (Assume that water evaporates but that the salt does not.) 67 mL of a 0.21 g/mL sugar solution is added to 402 mL of solvent. What is percentage (m/v) of sugar in the new solution? What is the % (v/v) of NaCl in a solution prepared by dissolving 25.5g of NaCl in 49 mL of water (densities: water = 1 g/mL, NaCl = 2g/mL) A 125 mL sample of a solution of ZnCl2 was diluted with water to 250 mL. A 50.0 mL sample of the dilute solution was found to contain 0.05 moles of ZnCl2. What was the concentration of ZnCl2 in the original undiluted solution?

Sample Questions This table indicates the sizes of the bands observed following restriction digests of a plasmid; Draw a possible map which is in agreement with these results. Note: No two restriction sites can occupy the same position and all restriction sites are separated by at least 0.5 Kbp. Indicate the position of each restriction site relative to the origin (EcoRI site). Enzymes Fragment sizes (Kbp) BamHI 9 EcoRI HindIII 2 and 7 PstI 5 and 4 BamHI + EcoRI 4.5 BamHI + HindIII 1.5, 2, and 5.5 BamHI + PstI 1.5, 2.5 and 5 EcoRI + HindIII 1, 2 and 6 EcoRI + PstI 2, 3 and 4 HindIII + PstI 1, 3 and 4

Sample Questions Following a separation on an agarose gel, a 12 Kbp fragment digested with BamHI resulted in three bands corresponding to migration distances of 7, 3, and 2Kbp. The same fragment digested with EcoRI resulted in only one band with a migration distance corresponding to 6Kbp. Draw all possible maps which would be in agreement with the banding pattern observed. Note: Do not include mirror maps. For example A-B-C and C-B-A would be considered mirror maps and thus the same. Indicate the positions of each restriction site relative to the origin. Label each of your maps alpha numerically (A, B, C, D, etc.)

Sample Questions Fill in the blanks to complete the following sequences in order to generate palindromes which would represent potential restriction sites. (Note: each blank represents a single nucleotide) 5' GC ___ A___ C 5' GGT ___ ___ ___ 5' ___ CTGC ___ GA 5' GGCT___GC ___ The enzyme FraII recognizes and cleaves the sequence C▼NNATNNG. What would be the average fragment length following the digestion of genomic DNA?

Sample Questions A restriction enzyme analysis of a recombinant plasmid is shown below. The first lane represents the undigested recombinant plasmid (UD). Lanes 2-4 represent BamHI, HindIII and PstI digested recombinant plasmid. The last lane represents the vector alone digested with HindIII. The numbers besides each band represent the approximate sizes in kilobase pairs. Which lane shows evidence of a partial digest? Indicate which bands are intermediate products by their size. How many sites remain to be digested in the intermediate products identified? How many times does Pst I cut within the recombinant plasmid?  How many times does HindIII cut within the BamH I fragment?

Sample Questions A PCR reaction was done to amplify a 500 bp sequence from 10 pg of the following single stranded 100 Kb template. The following primers were used: A: ATGACGAAG and B: ACCATCAGCA Which of the two primers is the forward primer? How many cycles would be required to obtain one microgram of the desired product? 3’ -••••••••••TGGTAGTCGTGC••••••••••GAAGCAGTA ••••••••••-5’ 1 │ 10000 10500 100000

Sample Questions You have a DNA solution with an absorbance at 260nm of 0.15. 0.4mL of this DNA solution is added to 0.6mL of water. What is the new concentration of DNA in mg/mL? Ans. 0.003 The concentration of a salt solution is 0.38% (m/v). 124 mL of this solution is placed in a beaker and allowed to evaporate until 92 mL remains. What is the new salt concentration in mg/ml? (Assume that water evaporates but that the salt does not.) Ans. 5.1 mg/mL 67 mL of a 0.21 g/mL sugar solution is added to 402 mL of solvent. What is percentage (m/v) of sugar in the new solution? Ans. 3% What is the % (v/v) of NaCl in a solution prepared by dissolving 25.5g of NaCl in 49 mL of water (densities: water = 1 g/mL, NaCl = 2g/mL) Ans. 20.64% NaCl A 125 mL sample of a solution of ZnCl2 was diluted with water to 250 mL. A 50.0 mL sample of the dilute solution was found to contain 0.05 moles of ZnCl2. What was the concentration of ZnCl2 in the original undiluted solution? Ans. 2M

Sample Questions This table indicates the sizes of the bands observed following restriction digests of a plasmid; Draw a possible map which is in agreement with these results. Note: No two restriction sites can occupy the same position and all restriction sites are separated by at least 0.5 Kbp. Indicate the position of each restriction site relative to the origin (EcoRI site). Enzymes Fragment sizes (Kbp) BamHI 9 EcoRI HindIII 2 and 7 PstI 5 and 4 BamHI + EcoRI 4.5 BamHI + HindIII 1.5, 2, and 5.5 BamHI + PstI 1.5, 2.5 and 5 EcoRI + HindIII 1, 2 and 6 EcoRI + PstI 2, 3 and 4 HindIII + PstI 1, 3 and 4

Sample Questions Following a separation on an agarose gel, a 12 Kbp fragment digested with BamHI resulted in three bands corresponding to migration distances of 7, 3, and 2Kbp. The same fragment digested with EcoRI resulted in only one band with a migration distance corresponding to 6Kbp. Draw all possible maps which would be in agreement with the banding pattern observed. Note: Do not include mirror maps. For example A-B-C and C-B-A would be considered mirror maps and thus the same. Indicate the positions of each restriction site relative to the origin. Label each of your maps alpha numerically (A, B, C, D, etc.)

Sample Questions Fill in the blanks to complete the following sequences in order to generate palindromes which would represent potential restriction sites. (Note: each blank represents a single nucleotide) 5' GC ___ A___ C GCTAGC 5' GGT ___ ___ ___ GGTACC 5' ___ CTGC ___ GA TCTGCAGA 5' GGCT___GC ___ GGCTAGCC The enzyme FraII recognizes and cleaves the sequence C▼NNATNNG. What would be the average fragment length following the digestion of genomic DNA? 4096

Sample Questions A restriction enzyme analysis of a recombinant plasmid is shown below. The first lane represents the undigested recombinant plasmid (UD). Lanes 2-4 represent BamHI, HindIII and PstI digested recombinant plasmid. The last lane represents the vector alone digested with HindIII. The numbers besides each band represent the approximate sizes in kilobase pairs. Which lane shows evidence of a partial digest? Indicate which bands are intermediate productsrmediate products identified? Indicate the intermediate products by their size. PstI 15, 12, and 5 How many sites remain to be digested in the intermediate products identified? 15(2), 12(1) and 5(1) How many times does Pst I cut within the recombinant plasmid?  3 times How many times does HindIII cut within the BamH I fragment? Twice

Sample Questions A PCR reaction was done to amplify a 500 bp sequence from 10 pg of the following single stranded 100 Kb template. The following primers were used: A: ATGACGAAG and B: ACCATCAGCA Which of the two primers is the forward primer? ATGACGAAG How many cycles would be required to obtain one microgram of the desired product? Initial = 0.5/100 X 10pg = 0.05pg of sequence of interest (single stranded) 3 cycles required to obtain double stranded product = 0.1pg 1 X 106pg = 0.1pg(2n) n= 23 5’ -••••••••••TGGTAGTCGTGC••••••••••GAAGCAGTA ••••••••••-3’ 1 │ 10000 10500 100000