1. 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

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

9. Midterm Breakdown Problem 3: 5 questions Restriction mapping
Southern analysis

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

11. Sample Questions
You have a DNA solution with an absorbance at 260nm of mL 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?

12. 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

13. 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.)

14. 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?

15. 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?

16. 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

17. Sample Questions
You have a DNA solution with an absorbance at 260nm of mL of this DNA solution is added to 0.6mL of water. What is the new concentration of DNA in mg/mL? Ans
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 % 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

18. 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

19. 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.)

20. 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

21. 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

22. 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