Post-Lab Assignment 2

CBE580 – Fall 2017

Name: Ryan Fitrian Sofwan Fauzan

Group 7
Group Members: Michael Joseph, Minsuk Song
Date: 9/8/2017

Objectives:

- To find out the relationship between cell concentration and optical density of cell

suspension.

- To do restriction digest of the plasmids using restriction enzymes.

1. Relationship between Optical Density and Cell Concentration from Serial Dilution Plates

and Optical Density of Cell

a. After performing the serial dilution and plating, the plates were checked and the number of

formed colonies were counted. The result is shown on Table 1 under the “Cell count” header.

Table 1. Cell count on the dilution plates for E. coli cells at 10-4, 10-5 and 10-6 dilution. TMTC
stands for “too many too count” and N/A means “not available” as the cell count is outside the
range of 30-200 colonies/plate.

Dilution Cell count Cell concentration (cells/mL)

10-4 TMTC N/A
TMTC N/A
10-5 114 1.14x109
116 1.16x109
-6
10 17 N/A
15 N/A
b. From the cell count data from Table 1, the only sample that produced colonies that are between

30-200 is the 10-5 dilution samples. Therefore, the cell concentration of the original cell

suspension is calculated from 10-5 dilution samples using the following equation:

# 𝐶𝐹𝑈𝑠 𝑏𝑎𝑐𝑡𝑒𝑟𝑖𝑎 𝑐𝑜𝑢𝑛𝑡𝑒𝑑

= (1)
𝑚𝐿 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 ∗ 𝑣𝑜𝑙𝑢𝑚𝑒 𝑖𝑛 𝑚𝑙 𝑎𝑝𝑝𝑙𝑖𝑒𝑑 𝑡𝑜 𝑝𝑙𝑎𝑡𝑒

Sample calculation (dilution 10-5, plate 1)

# 𝐶𝐹𝑈𝑠 114
= = 1.14𝑥109 𝑐𝑒𝑙𝑙𝑠/𝑚𝐿
𝑚𝐿 10−5 ∗ 0.01 𝑚𝐿

The result for the second plate of 10-5 dilution is 1.16x109 cells/mL, and the average value is

1.15x109 cells/mL.

c. During Lab 1 experiment, several cell suspensions were made with various dilution factors for

optical density (OD) measurement purposes. Table 2 shows the dilution factors of the E. coli cell

suspension.

Table 2. E. coli cells suspension dilutions. Each dilution was made by mixing different volume of
original cell suspension and LB-Amp media.
Sample % Cell Suspension Volume of Volume of LB-
Cells Amp Media
1 100 1.0 ml 0 ml
2 80 0.8 ml 0.2 ml
3 60 0.6 ml 0.4 ml
4 40 0.4 ml 0.6 ml
5 30 0.3 ml 0.7 ml
6 20 0.2 ml 0.8 ml
7 10 0.1 ml 0.9 ml
8 0 (blank) 0.0 ml 1.0 ml

After knowing the concentration of the original cell suspension (1.15x109 cells/mL), the data

from Table 2 can be converted from % cell suspension into concentration in cells/mL. The

converted data are shown on Table 3.

 Table 3. E. coli cells suspension concentrations. As the concentration of the original cell
suspension is known, the %cell suspension values are converted to cell concentration (cells/mL)
Sample % Cell Suspension Cell concentration
(cells/mL)
1 100 1.15x109
2 80 0.92x109
3 60 0.69x109
4 40 0.46x109
5 30 0.35x109
6 20 0.23x109
7 10 0.12x109
8 0 (blank) 0

d. After preparing the cell suspensions, the optical density (OD) of the cell suspensions were then

measured. Table 4 shows the OD data of the cell suspensions.

Table 4. Optical density data for E. coli cell suspensions. OD measurement was done in triplicate
for each sample, including the sample without cells (blanks).
Sample Cell concentration Optical Density
(cells/mL) A B C
1 1.15x109 0,4858 0,43 0,4322
2 0.92x109 0,2941 0,3693 0,3733
3 0.69x109 0,299 0,2864 0,2798
4 0.46x109 0,1991 0,2066 0,2064
5 0.35x109 0,1638 0,1605 0,1829
6 0.23x109 0,1342 0,1324 0,1316
7 0.12x109 0,0826 0,0952 0,0831
8 0 (blank) 0,0395 0,0589 0,0393

The average value of the blanks is 0.0459, which was then subtracted to each data point for the

samples with the cells (the blank value is then plotted as 0,0). The adjusted values of the OD data

are shown on Table 5.

 Table 5. Adjusted value of OD after subtracted with average blank. For a more accurate results,
the average of OD of the blanks was subtracted to each data point of OD with cells.
Sample Cell concentration Optical Density
(cells/mL) A B C
1 1.15x109 0.4399 0.3841 0.3863
2 0.92x109 0.2482 0.3234 0.3274
3 0.69x109 0.2531 0.2405 0.2339
4 0.46x109 0.1532 0.1607 0.1605
5 0.35x109 0.1179 0.1146 0.137
6 0.23x109 0.0883 0.0865 0.0857
7 0.12x109 0.0367 0.0493 0.0372
8 0 0 0 0

The data from Table 5 were then plotted with cell concentration (109 cells/mL) as the y-axis and

the optical density as the x-axis, as shown on Figure 1. Despite some scatters between each

replicate, the overall trend shows a linear correlation between the two variables. The squared R

value is also good at 0.9768. The equation obtained from the plot is as follows:

Y = 2.8803 x 109X + 1.1 x 106 (1)

Where Y is cell concentration (cells/mL) and X is optical density (unitless).

1.4
Cell concentration (109cells/mL)

y = 2.8803x + 0.0011
1.2 R² = 0.9768
1.0

0.8

0.6

0.4

0.2

0.0
0 0.1 0.2 0.3 0.4 0.5
Optical density

Figure 1. Optical density vs. cell concentration (109 cells/mL) of the dilution samples. R2 value of 0.9768
shows a linear correlation between optical density and cell concentration.
 Using the Data Analysis on Excel, the confidence interval for the slope is 2.8803 (±0.195) x 109

and for the intercept is 1.1 (±43.3) x 106.

e. During Lab 1 experiment, there were plates streaked with pGEM-luc and pUC-19 E. coli cells.

One colony from each plate were then grown in a liquid LB-Amp media in a flask. During Lab 2

experiment, the OD of both liquid cultures were measured as shown in Table 6.

Table 6. OD data for the pGEM-luc and pUC19 E. coli flask cultures

Culture OD-1 OD-2 OD-3

pGEM-luc 0.6349 0.6717 0.6572
pUC-19 0.59 0.6255 0.618
Blank 0.0389 0.0395 0.0391

The average OD of the blanks was then deducted to the OD of pGEM-luc and pUC-19 cells, and

the final data are shown in Table 7. The average OD of each cell were then calculated.

Table 7. Final OD data for the pGEM-luc and pUC19 E. coli flask cultures
Culture OD-1 OD-2 OD-3 Average OD
pGEM-luc 0.5957 0.6325 0.6180 0.6154
pUC-19 0.5508 0.5863 0.5788 0.5720

Using equation (1), the cell concentration of the liquid culture can be estimated as follows:

 pGEM-luc: Y=2.8803 x 109 * (0.6154) + 1.1 x 106 = 1.77x109 cells/mL

 pUC-19 : Y=2.8803 x 109 * (0.5720) + 1.1 x 106 = 1.65x109 cells/mL
f. The cell concentration of the original cell suspension is 1.15 x 109 cells/mL, as calculated from

the data obtained during the serial dilutions and plating experiments in Lab 1. In addition, the

optical density (OD) data measured from the diluted suspensions were then plotted against the

cell concentration. The plot showed a linear correlation with a R2 value of 0.9768 with the

equation (1) below:

Y = 2.8803 x 109 X + 1.1 x 106 (1)

Y = cell concentration, cells/ml

X = OD of cell suspension, unitless

Therefore, optical density value can be used as a way to indirectly measure the concentration of a

cell suspension. Finally, when equation (1) was used to estimate the concentrations of flask

cultures of pGEM-luc and pUC-19, the results are 1.77x109 and 1.65x109 cells/mL respectively.

2. pGEM-luc and pUC19 Resctriction Sites for Bam HI and Sac I

The restriction sites for SacI and BamHI are as follows:

 SacI restriction site: GAGCT/C

 BamHI restriction site: G/GATCC

From https://www.neb.com/tools-and-resources/interactive-tools/dna-sequences-and-maps-tool

and https://www.ncbi.nlm.nih.gov/nuccore/X65316.2?report=fasta, the sequences of pUC-19 and

pGEM-luc plasmids are obtained, respectively. SacI and BamHI restriction sites are in bold and

10 pt font in both plasmid sequences.

pUC-19 plasmid (2686 base pairs)

TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGG
GAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGT
ACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAG
GCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGA
TTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTC GAGCT/CGGTACCCGGG/GA
TCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACA
ATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGC
GCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCG
TATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGG
CGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGT
AAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGC
GAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACC
GGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTT
CGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCC
GGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTT
CTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAA
AGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAA
AAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGT
CATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAA
CTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCC
CCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGC
TCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCT
ATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGT
GTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAA
AAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCAT
AATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCG
GCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAA
CGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTC
AGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACG
GAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGA
ATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATC
ATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC.

If pUC-19 plasmid is cut with both SacI and BamHI restriction enzymes, the shorter fragment

would have 11 base pairs and the longer fragment would have 2675 base pairs.

pGEM-luc (4931 base pairs)

GGGCGAATTGGCCAAGTCGGCCGAGCT/CGAATTCGTCGACCTCGAGGCCTCGGAGGATTACAATAGCTAAGAATTTCGTCATC
GCTGAATACAGTTACATTTTACAATTTGGACTTTCCGCCCTTCTTGGCCTTTATGAGGATCTCTCTGATTTTTCTTGCGTCGAGTTTT
CCGGTAAGACCTTTCGGTACTTCGTCCACAAACACAACTCCTCCGCGCAACTTTTTCGCGGTTGTTACTTGACTGGCGACGTAATC
CACGATCTCTTTTTCCGTCATCGTCTTTCCGTGCTCCAAAACAACAACGGCGGCGGGAAGTTCACCGGCGTCATCGTCGGGAAGAC
CTGCCACGCCCGCGTCGAAGATGTTGGGGTGTTGTAACAATATCGATTCCAATTCAGCGGGGGCCACCTGATATCCTTTGTATTTA
ATTAAAGACTTCAAGCGGTCAACTATGAAGAAGTGTTCGTCTTCGTCCCAGTAAGCTATGTCTCCAGAATGTAGCCATCCATCCTT
GTCAATCAAGGCGTTGGTCGCTTCCGGATTGTTTACATAACCGGACATAATCATAGGTCCTCTGACACATAATTCGCCTCTCTGAT
TAACGCCCAGCGTTTTCCCGGTATCCAGATCCACAACCTTCGCTTCAAAAAATGGAACAACTTTACCGACCGCGCCCGGTTTATCA
TCCCCCTCGGGTGTAATCAGAATAGCTGATGTAGTCTCAGTGAGCCCATATCCTTGTCGTATCCCTGGAAGATGGAAGCGTTTTGC
AACCGCTTCCCCGACTTCTTTCGAAAGAGGTGCGCCCCCAGAAGCAATTTCGTGTAAATTAGATAAATCGTATTTGTCAATCAGAG
TGCTTTTGGCGAAGAATGAAAATAGGGTTGGTACTAGCAACGCACTTTGAATTTTGTAATCCTGAAGGGATCGTAAAAACAGCTC
TTCTTCAAATCTATACATTAAGACGACTCGAAATCCACATATCAAATATCCGAGTGTAGTAAACATTCCAAAACCGTGATGGAAT
GGAACAACACTTAAAATCGCAGTATCCGGAATGATTTGATTGCCAAAAATAGGATCTCTGGCATGCGAGAATCTGACGCAGGCAG
TTCTATGCGGAAGGGCCACACCCTTAGGTAACCCAGTAGATCCAGAGGAATTCATTATCAGTGCAATTGTTTTGTCACGATCAAAG
GACTCTGGTACAAAATCGTATTCATTAAAACCGGGAGGTAGATGAGATGTGACGAACGTGTACATCGACTGAAATCCCTGGTAAT
CCGTTTTAGAATCCATGATAATAATTTTCTGGATTATTGGTAATTTTTTTTGCACGTTCAAAATTTTTTGCAACCCCTTTTTGGAAAC
AAACACTACGGTAGGCTGCGAAATGTTCATACTGTTGAGCAATTCACGTTCATTATAAATGTCGTTCGCGGGCGCAACTGCAACTC
CGATAAATAACGCGCCCAACACCGGCATAAAGAATTGAAGAGAGTTTTCACTGCATACGACGATTCTGTGATTTGTATTCAGCCC
ATATCGTTTCATAGCTTCTGCCAACCGAACGGACATTTCGAAGTATTCCGCGTACGTGATGTTCACCTCGATATGTGCATCTGTAA
AAGCAATTGTTCCAGGAACCAGGGCGTATCTCTTCATAGCCTTATGCAGTTGCTCTCCAGCGGTTCCATCCTCTAGAGGATAGAAT
GGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCATTTG/GATCCGGGCCCTCTAGATGCGGCCGCATGCATAAGCTTGAGTA
TTCTATAGTGTCACCTAAATAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACA
ACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCC
CGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGC
TCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACG
GTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCG
CGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGAC
AGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGT
CCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGC
TGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACAC
GACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGT
GGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAG
CTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCT
CAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATC
AAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACA
GTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGA
TAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATC
AGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGC
CGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTC
GTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCT
CCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTG
TCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGC
TCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGC
GAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACT
TTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATA
CTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAA
AATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCT
ATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGA
GACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGG
CTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGA
AAATACCGCATCAGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGC
CAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGG
GCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCGCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCAT
CGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTC
AACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAA
TTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTG
CGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGT
CACGACGTTGTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATA.

If pGEM-luc plasmid is cut with both SacI and BamHI restriction enzymes, the shorter fragment

would have 1730 base pairs and the longer fragment would have 3201 base pairs.

Table 8 shows the summary for the number of base pairs of DNA fragments of the plasmids.

Table 8. Number of base pairs of DNA fragments of pUC-19 and pGEM-luc plasmids cut by SacI and
BamHI restriction enzymes.

Plasmid Length of shorter DNA fragment Length of longer DNA fragment

(bps) (bps)
pUC-19 11 2675
pGEM- 1730 3201
luc

References
https://www.neb.com/tools-and-resources/interactive-tools/dna-sequences-and-maps-tool
(accessed 9/13/2017).
https://www.ncbi.nlm.nih.gov/nuccore/X65316.2?report=fasta (accessed 9/13/2017).