Amplification and restriction d

Name :

ID number:

Course name: Biotechnology

Course Code: BIOT3113

Title: Amplification of LacZ gene fragment and restriction digestion of pRY121 plasmid from

E. coli.

Date: November 19, 2022

Aim: To amplify the lacZ gene fragment by PCR and to digest the pRY121 plasmid using

several restriction enzymes and conducting an agarose gel electrophoresis.

Abstract:

In the experiment, the aim was to amplify the lacZ gene fragment by PCR, use gel

electrophoresis to find the size of the PCR product, also to digest the pRY121 plasmid using

several restriction enzymes namely HindIII, PstI, BamHI, and EcoRI. The determination of the

band sizes was done by conducting an agarose gel electrophoresis. The methos consisted of

adding 2 µL of the sample DNA to the PCR tube containing the master mix for each of the

enzymes and after PCR the gel electrophoresis was done. The results of this show that the lacZ

gene was successfully amplified as the band size observed was similar to the band size expected.

The results of the restriction digestion showed that for HindIII there was possibly DNA

secondary structure contamination present resulting in an extra band, The EcoRI and PstI gels

showed extremely large deviations to the expected results and observed band sizes. The band

size of BamHI could not be properly reported. To conclude the Amplification of the lacZ gene

was successful however, the restriction digestion consisted of a lot of discrepancies, but was still

deemed successful.
Method:

As seen in BIOT3113 Lab Manual with the following changes :

Volume Changes

Table 1. The volumes of each reagent added to make the master mix.

BamHI and PstI EcoRI and HindIII

x1 x32 x1 x32

H2O 5.5 µL 176 µL 6.5 µL 208 µL

BSA 1 µL 32 µL - -

Buffer 1 µL 32 µL 1 µL 32 µL

Enzyme 0.5 µL 16 µL 0.5 µL 16 µL

DNA 2 µL N/A 2 µL N/A

Total 10 µL 8 µL each 10 µL 8 µL each

 Results:

Diagram 1. Gel electrophoresis of the amplification of lacZ gene DNA from plasmid pRY121.

100 bp DNA ladder

Sample St TC 600 bp

Positive Control
 SAMPLE

A1
100 bp DNA POSITION
Ladder
SAMPLE

A2
TA POSITION

SAMPLE

A3
SS G POSITION

SAMPLE

A4
ST TC POSITION

SAMPLE

A5
RC RB POSITION

SAMPLE

A6
SG POSITION

SAMPLE

A7
DR SA POSITION

SAMPLE

A8
JN RG POSITION

SAMPLE

A9
BD RI POSITION

SAMPLE

A10
Positive POSITION
Control
SAMPLE

A11
POSITION

SAMPLE

A12
POSITION

SAMPLE

A13
POSITION

SAMPLE

A14
POSITION
Table 2. The Sample positions, ladders and lane assignment for the agarose electrophoresis gel.
Diagram 2. Electrophoretogram of digestion of E. coli plasmid pRY121 using enzyme PstI

Sample TI, BF - PstI

1 kb DNA ladder

Lamda HindIII DNA

ladder
 SAMPLE

A1
1 Kb DNA POSITION
Ladder
SAMPLE

A2
KMR POSITION
EcoRI
SAMPLE

A3
PstI POSITION

SAMPLE

A4
HindIII POSITION

SAMPLE

A5
BamHI POSITION

SAMPLE

A6
TI BF POSITION
EcoRI

SAMPLE

A7
HindIII POSITION

SAMPLE

A8
BamHI POSITION

SAMPLE

A9
PstI POSITION

SAMPLE

A10
JHCC POSITION
EcoRI

SAMPLE

A11
PstI POSITION

SAMPLE

A12
HindIII POSITION

SAMPLE

A13
BamHI POSITION

Diagram 3. Electrophoretogram of digestion of E. coli plasmid pRY121 using enzyme BamHI

SAMPLE

A14
Lambda POSITION
Table 3. The Sample positions, ladders and lane assignment for the agarose electrophoresis gel.

HindIII
Digest
 SAMPLE

A1
EC POSITION
PstI
SAMPLE

A2
EcoRI POSITION

SAMPLE

A3
HindIII POSITION

SAMPLE

A4
BamHI POSITION

SAMPLE

A5
RGSO POSITION
PstI
SAMPLE

A6
EcoRI POSITION

SAMPLE

A7
HindIII POSITION

SAMPLE

A8
BamHI POSITION

SAMPLE

A9
1 Kb DNA POSITION
Ladder
SAMPLE

A10
Lambda POSITION
HindIII Digest
SAMPLE

A11
KC RD POSITION
HindIII
SAMPLE

A12
PstI POSITION
Ladder

Table 4. The Sample positions, ladders and lane assignment for the agarose electrophoresis gel.

SAMPLE

A13
BamHI POSITION
1kb DNA ladder

SAMPLE
Sample EC - BamHI

A14
EcoRI
Lambda HindIII DNA

POSITION
 SAMPLE

A1
1 Kb DNA POSITION
Ladder
SAMPLE

A2
Lambda POSITION
HindIII
SAMPLE

A3
AA AG POSITION
BamHI
SAMPLE

A4
EcoRI POSITION

SAMPLE

A5
HindIII POSITION

SAMPLE

A6
PstI POSITION

SAMPLE

A7
SSSBTGK POSITION
W
PstI
SAMPLE

A8
HindIII POSITION

SAMPLE

A9
EcoRI POSITION

SAMPLE

A10
BamHI POSITION

SAMPLE

A11
DM DP POSITION
KW
EcoRI
SAMPLE
ladder

A12
HindIII

BamHI POSITION
Diagram 4. Electrophoretogram of digestion of E. coli plasmid pRY121 using enzyme HindIII

Table 5. The Sample positions, ladders and lane assignment for the agarose electrophoresis gel.
SAMPLE

A13
HindIII POSITION
1 kb DNA ladder
Lambda HindIII DNA

SAMPLE

A14
Sample DM, DP, KW -

PstI POSITION
Diagram 5. Electrophoretogram of digestion of E. coli plasmid pRY121 using enzyme EcoRI

Sample SG – EcoR1

1 kb DNA Ladder

Lambda HindIII DNA

Ladder

Table 6. The Sample positions, ladders and lane assignment for the agarose electrophoresis gel.
 SAMPLE POSITION

SAMPLE POSITION

SAMPLE POSITION

SAMPLE POSITION

SAMPLE POSITION
POSITION

POSITION

POSITION

POSITION

POSITION

POSITION

POSITION

POSITION

POSITION
SAMPLE

SAMPLE

SAMPLE

SAMPLE

SAMPLE

SAMPLE

SAMPLE

SAMPLE

SAMPLE
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14

1 Kb DNA

Lambda
SM ML

Ladder
BamHI

BamHI

BamHI
HindIII

HindIII

HindIII

HindIII
TRAM
EcoRI

EcoRI

EcoRI
PstI

PstI

PstI
SG
Restriction Enzyme Expected Sizes (bp) Observed Sizes (bp)

PstI 8110 BP ≈9800

2940BP ≈3100

450BP

EcoRI 3530BP ≈<10,000

2880BP ≈3730

2830BP ≈3130

2260BP ≈2820

HindIII 6630BP ≈9230

2770BP ≈2810

2100BP ≈1910

≈1430

BamHI 11500BP ≈<10,000

Table 7: The expected and observed band sizes for the 4 restriction enzymes used to

digest the pRY121 plasmid

Calculations:

1. State original DNA concentration based on previous quantification results

The concentration of the diluted DNA sample was 0.4 µg/µl based on previous quantification

results.

There dilution factor was 1:100

To obtain the original concentration we multiply by the dilution factor

100 x 0.4 µg/µl = 40 µg/µl

2. For PCR, calculations to show how that DNA should be diluted to obtain a concentration

of 1µg/ml in 4µl.

A 1:10 dilution, by transferring 1 µL of DNA to 9 µL of diluent to get a

concentration of 4 µg/µl. Then remove 1 µl from that solution and add to 3 µl to give a

dilution of 1µg/ml in 4µl.

3. Sample calculation of sizes of expected fragments for at least one restriction digestions

except BamHI.

Fragment sizes for EcoRI

 11500 bp -11380 bp + 2760 bp = 2880 bp

 1380 bp-9120 bp = 22600 bp

 9120 bp-5590 bp = 3530 bp

 5590 bp – 2760 bp = 2830 bp

Discussion:

lacZ Amplification

The main aim in this section of the experiment was to amplify the lacZ gene that is found

in the plasmid pRY121 present in E. coli. The sample in lane A4 was used to calculated the size

of the gene after amplification. This was observed on the agarose gel electrophoresis as 600 bp.

The size of the lacZ gene is 562 bp and the observed result was not very far off. This indicates

the successful amplification of the lacZ gene.

The limitations in PCR has to do with the primer specificity, in order to obtain a specific

primer, the knowledge of the target sequence is required. A source of error in this experiment is

that PCR is highly sensitive and will amplify any DNA present in the sample including

contaminants.

Restriction Digestion

For the PstI restriction enzyme, there are 3 expected band sizes with sizes 450 bp, 2940

bp and 8110 bp. The bands observed in lane A9 were inaccurate as only two bands were

observed with sizes 9900 bp and 3150 bp, these values are largely different from the expected

values. Incomplete digestion could be implied or insufficient amounts of DNA utilised.

The restriction using BamHI was expected to cut the plasmid resulting in a size of 11,

500 bp. This fragment is unable to fit on the 1 kb ladder, however in lanes A4 there was a band

with size greater then 10000 which may have been the expected fragment, however without the

use of a different ladder it is only an assumption.

The HindIII digests sample showed bands at 14300bp, 1910bp, 2810bp, and 9230bp.

The expected bands were at 2100 bp, 27770 bp, and 6630 bp. There were more bands observed
than unexpected this could have been due to binding between strands in which case a

denaturation is required to separate the binding. There was also poor band separation observed

on this gel which could have been due to insufficient run time. The sizes observed vs expected

were also larger, this would indicate that something other than the plasmid, genomic DNA, was

included in the sample as a contaminant.

The EcoRI enzyme, is expected to have 4 distinct bands with sizes, 2260 bp, 2830 bp,

2880 bp and 3530 bp. There were 4 observed bands however the sizes were not similar. The

sizes observed were 2800 bp, 3100 bp, 3700 bp, and < 1000 bp. The enzyme may have been

operating properly however the contamination in the DNA as well as errors in primer binding

could have caused these deviations.

Conclusion:

In conclusion, the amplification of the lacZ gene was successful and the restriction digestion of

plasmid pRY121 contained several discrepancies but deemed to be successful.

Questions:

1. Suppose your PCR reaction yielded multiple products in addition to the one you

desired; without using different primers, what changes would you make to the

reaction to increase specificity ?

To increase primer specificity you can adjust the length of the primer. A desired length is

between 23 and 26 nucleotides, with a high annealing temperature around 65°C to reduce the risk

of error in primer binding. Specificity can also be improved by having an amplicon length of

100-200 nucleotides and lowering the elongation timings of the PCR procedure. Additionally,

PCR polymerases with “hot -start” capabilities will reduce errors.

2. If a PCR reaction did not yield any products where one was expected, suggest four

things you could do to alter to obtain a product. Explain the rationale behind each

suggestion.

- The use of a positive control to ensure the presence and function of the master mix

components, if all components are not present the desired reaction will not occur.

- PCR may be inhibited by the final volume and concentration of the template material

which may be too low or too high.

- The most suitable primers should be used to ensure DNA binding, and prevent unwanted

binding and formation of dimers.

- The purity and compatibility of the primer and target sequence should be tested as

contaminants may hinder the PCR reaction.

 3. Prepare a reaction mixture for a restriction digest containing components from the

following stock solutions: DNA (2 mg/ml), BSA (10x reaction buffer) (10X and

BamHI (10 U//uI). Indicate the minimum total reaction volume required for

digesting 10ug of plasmid DNA and the order in which these reagents should be added.

2mg/ml = 2ug/ul

The Final volume is 20μl

The amount of [BSA] required

= 1x. Since the stock is 10x, then a 1 in 10 dilutions is carried out. The volume of BSA to be

added is 2μl.

The amount of reaction buffer required

= 1x however, 10x is required to be added to the stock (buffer is always 10% of the stock

volume) a 1:10 dilution takes place and 2μl of the buffer is added.

The volume of enzyme required

= 2μl, a 1:5 dilutions and 1μl of enzyme would be added.

DNA Required

= 2mg/ml = 2μl/μg

So 1 μl = 2μg

xμl= 10μg

So the amount of DNA required would be = 5μl. 10μl of distilled water will then be added to

make it up 20μl.
 4. Calculate the expected sizes of the DNA fragments from the following digest of

pRY121:

a. SacI

One restriction site, 4630 bp

b. HindIII and PstI

0 and 210 = 210

0 bp and 210 bp which is equal to 210bp

1000 b p(HindIII) – PstI 210bp = 790bp

2780 bp (HindIII) – 1000 (Pst1) = 1780bp

8230 bp (HindIII) -2780 bp( Pst1) =5540bp

9400 bp (HindIII) – 8320bp (Pst1) = 1080bp

11260 bp (HindIII) -9400(Pst1) = 1860bp

c. SmaI and EcoRI

Cuts at 960bp, 2760bp, 2830bp, 3530bp, and 2260bp

Fragments: 1080bp, 1800bp,2830bp, 3530bp and 2260BP

d. XboI

No restriction site present on the plasmid

 5. What would be the effect of plasmid function of subcloning (“inserting”) a 1.0-kb

gene fragment into:

a. The SacI site in pRY121?

Synthesis if beta-galactosidase will be stopped. It is responsible for converting galactosidase

into monosaccharides.

b. The SmaI site in the pRY121?

The plasmid is expected to be cut once where there are no functional genes, it is expected that

any effect will be negligible. The tetracycline resistance gene's open reading frame may be

relocated further down of the plasmid.

c. The PstI site in URA3 gene in pRY121?

Uracil and Uridines are necessary and are synthesized using the enzyme, orotidine 5’ phosphate.

The synthesis of this enzyme will be stopped.

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