You are on page 1of 24

University “Alexandru Ioan

Cuza” of Iași

Erasmus+ stage practice June-July 2017

Coordinator: Student:

Natalia Tomás Marques Mădălina-Bianca Bujder

1
Table of contents

1.Introduction....................................................................3
1.1 Cloning a Specific Gene ...........................................4
1.2 Choosing a cloning vector ........................................4
1.3 Plasmids ....................................................................4
1.4 Polymerase chain reaction (PCR) .............................5
1.5 Steps for Using Bacteria and Plasmids to Clone
Genes ..............................................................................8
2. Material and methods ..................................................10
2.1 Recombinant plasmid pQE-10 ................................10
2.2 Transformation of DH5α E.colli cells.....................11
2.3 Electrophoresis Gel .................................................13
2.4 Purification of gels from agarose of the MiniPreps
using Megaquick-spinTM Total Fragment DNA
Purification Kit .............................................................16
3. Results.........................................................................23
4. Bibliography ...............................................................24

2
1.Introduction

All living organisms have common characteristics such as replication, nutrition, growing
and interaction with their environment. An organism is composed of organs which perform
specific functions. Organs are made of tissues which are composed of aggregation of cells that
have similar functions. The cell is the basic unit of life in all living organisms and it has
molecules that have fundamental functions for life. Molecular biology is the study of these
molecules in the cell. Two of these molecules called proteins and nucleotides have fundamental
roles to sustain life. Proteins are the key components in everything related to life. DNA is made
of nucleotides and parts of DNA called genes code for proteins which perform all the
fundamental processes for living using biochemical reactions. Cells synthesize new molecules
and break large molecules into smaller ones using complex networks of chemical reactions
called pathways. Genome is the complete set of DNA of an organism which contain many genes.
A gene is the basic physical and functional unit of hereditary that codes for a protein which is a
large molecule made from a sequence of amino acids. Three critical molecules of life are
deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and proteins. A central paradigm in
molecular biology states that biological function is heavily dependent on the biological structure.

In the time that I spend working for two months in this stage practice I learned what is the
meaning of transformation cells. Transformation happens when bacteria take up DNA from the
environment and then convert the genes encoded by the DNA into a protein or trait that can be
observed. Bacteria have one circular genomic chromosome, but some bacteria have extra DNA
called plasmids. Foreign DNA can be inserted on a plasmid or onto the genomic chromosome.
Also that in molecular biology the ligation reaction it is commonly used for the insertion of
restriction enzyme-generated DNA fragments into vector. Also learning that PCR is based on
using the ability of DNA polymerase to synthesize new strand of DNA complementary to the
offered template strand. Because DNA polymerase can add a nucleotide only onto a preexisting
3'-OH group, it needs a primer to which it can add the first nucleotide. This requirement makes it
possible to delineate a specific region of template sequence that the researcher wants to amplify.
At the end of the PCR reaction, the specific sequence will be accumulated in billions of copies
(amplicons).

In the next pages, we first review about the plasmid that we will use (Recombinat
Plasmid pQE-10 with mCherry) and gene 1,8Cin and the protocols that we used for our purpose,
and that is the amplify this gene into the plasmid.

3
1.1 Cloning a Specific Gene

The foregoing descriptions are generic approaches to creating recombinant


DNA. However, a geneticist is interested in isolating and characterizing some
particular gene of interest, so the procedures must be tailored to isolate a specific
recombinant DNA clone that will contain that particular gene. The details of the
process differ from organism to organism and from gene to gene. An important
initial factor is the choice of an appropriate vector for the job at hand.

1.2 Choosing a cloning vector

Vectors must be relatively small molecules for convenience of manipulation. They


must be capable of prolific replication in a living cell, thereby enabling
the amplification of the inserted donor fragment. Another important requirement is
that there be convenient restriction sites that can be used for insertion of
the DNA to be cloned. Unique sites are most useful because then the insert can be
targeted to one site in the vector. It is also important that there be a mechanism for
easy identification and recovery of the recombinant molecule. There are numerous
cloning vectors in current use, and the choice between them often depends on the
size of the DNA segment that needs to be cloned. We will consider several
commonly used types.

1.3 Plasmids

Bacterial plasmids are small circular DNA molecules that are distinct from, as well
as additional to, the main bacterial chromosome. They replicate their DNA
independently of the bacterial chromosome. Many different types of plasmids have
been found in bacteria. The distribution of any one plasmid within a species is
generally sporadic; some cells have the plasmid, whereas others do not.

4
1.4 Polymerase chain reaction (PCR)

Polymerase chain reaction (PCR) is a technique used in molecular biology to amplify a


single copy or a few copies of a segment of DNA across several orders of magnitude, generating
thousands to millions of copies of a particular DNA sequence.

Principles
PCR amplifies a specific region of a DNA strand (the DNA target). Most PCR methods amplify
DNA fragments of between 0.1 and 10 (kbp), although some techniques allow for amplification
of fragments up to 40 kbp in size. The amount of amplified product is determined by the
available substrates in the reaction, which become limiting as the reaction progresses.
A basic PCR set-up requires several components and reagents including:

 a DNA template that contains the DNA target region to amplify


 a DNA polymerase, an enzyme that polymerizes new DNA strands; heat-resistant Taq
polymerase is especially common, as it is more likely to remain intact during the high-
temperature DNA denaturation process
 two DNA primers that are complementary to the 3’ (three prime) ends of each of the sense
and anti-sens strands of the DNA target (DNA polymerase can only bind to and elongate
from a double-stranded region of DNA; without primers there is no double-stranded
initiation site at which the polymerase can bind); specific primers that are complementary to
the DNA target region are selected beforehand, and are often custom-made in a laboratory or
purchased from commercial biochemical suppliers
 deoxynucleoside triphosphates, or dNTPs (sometimes called "deoxynucleotide
triphosphates"; nucleotides containing triphosphate groups), the building blocks from which
the DNA polymerase synthesizes a new DNA strand
 a buffer solution providing a suitable chemical environment for optimum activity and
stability of the DNA polymerase
 bivalent cations, typically magnesium (Mg) or manganese (Mn) ions; Mg2+ is the most
common, but Mn2+ can be used for PCR-mediated DNA mutagenesis, as a higher
Mn2+ concentration increases the error rate during DNA synthesis
 monovalent cations, typically potassium (K) ions;

Procedure
Typically, PCR consists of a series of 20–40 repeated temperature changes, called cycles,
with each cycle commonly consisting of two or three discrete temperature steps (see figure 4
below).The cycling is often preceded by a single temperature step at a very high temperature
(>90 °C (194 °F)), and followed by one hold at the end for final product extension or brief
storage. The temperatures used and the length of time they are applied in each cycle depend
on a variety of parameters, including the enzyme used for DNA synthesis, the concentration

5
of bivalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.
The individual steps common to most PCR methods are as follows:

 Initialization: This step is only required for DNA polymerases that require heat
activation by hot start PCR. It consists of heating the reaction chamber to a temperature
of 94–96 °C (201–205 °F), or 98 °C (208 °F) if extremely thermostable polymerases are
used, which is then held for 1–10 minutes.
 Denaturation: This step is the first regular cycling event and consists of heating the
reaction chamber to 94–98 °C (201–208 °F) for 20–30 seconds. This causes DNA
melting, or denaturation, of the double-stranded DNA template by breaking the hydrogen
bonds between complementary bases, yielding two single-stranded DNA molecules.
 Annealing: In the next step, the reaction temperature is lowered to 50–65 °C (122–
149 °F) for 20–40 seconds, allowing annealing of the primers to each of the single-
stranded DNA templates. Two different primers are typically included in the reaction
mixture: one for each of the two single-stranded complements containing the target
region. The primers are single-stranded sequences themselves, but are much shorter than
the length of the target region, complementing only very short sequences at the 3' end of
each strand.
It is critical to determine a proper temperature for the annealing step because
efficiency and specificity are strongly affected by the annealing temperature. This
temperature must be low enough to allow for hybridization of the primer to the strand,
but high enough for the hybridization to be specific, i.e., the primer should
bind only to a perfectly complementary part of the strand, and nowhere else. If the
temperature is too low, the primer may bind imperfectly. If it is too high, the primer
may not bind at all. A typical annealing temperature is about 3–5 °C below the Tm of
the primers used. Stable hydrogen bonds between complementary bases are formed
only when the primer sequence very closely matches the template sequence. During
this step, the polymerase binds to the primer-template hybrid and begins DNA
formation.

 Extension/elongation: The temperature at this step depends on the DNA polymerase


used; the optimum activity temperature for Taq polymerase is approximately 75–
80 °C (167–176 °F), though a temperature of 72 °C (162 °F) is commonly used with
this enzyme. In this step, the DNA polymerase synthesizes a new DNA strand
complementary to the DNA template strand by adding free dNTPs from the reaction
mixture that are complementary to the template in the 5'-to-3'
direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxy
group at the end of the nascent (elongating) DNA strand. The precise time required
for elongation depends both on the DNA polymerase used and on the length of the
DNA target region to amplify. As a rule of thumb, at their optimal temperature, most
DNA polymerases polymerize a thousand bases per minute. Under optimal
conditions (i.e., if there are no limitations due to limiting substrates or reagents), at
each extension/elongation step, the number of DNA target sequences is doubled.

6
With each successive cycle, the original template strands plus all newly generated
strands become template strands for the next round of elongation, leading to
exponential (geometric) amplification of the specific DNA target region.
The processes of denaturation, annealing and elongation constitute a single cycle.
Multiple cycles are required to amplify the DNA target to millions of copies. The
formula used to calculate the number of DNA copies formed after a given number of
cycles is 2n, where n is the number of cycles. Thus, a reaction set for 30 cycles results
in 230, or 1073741824, copies of the original double-stranded DNA target region.

 Final elongation: This single step is optional, but is performed at a temperature


of 70–74 °C (158–165 °F) (the temperature range required for optimal activity of
most polymerases used in PCR) for 5–15 minutes after the last PCR cycle to
ensure that any remaining single-stranded DNA is fully elongated.
 Final hold: The final step cools the reaction chamber to 4–15 °C (39–59 °F) for
an indefinite time, and may be employed for short-term storage of the PCR
products.

Fig.4 PCR Reaction

Sourse: http://lab.gentaur.com/pcr.htm

7
1.5 Steps for Using Bacteria and Plasmids to Clone Genes

Recombinant DNA molecules are only useful if they can be made to replicate
and produce a large number of copies. A typical gene-cloning procedure includes the
following steps:

Step 1: Isolation of two kinds of DNA.

 Bacterial plasmids and foreign DNA containing the gene of interest are
isolated.

--> ampR that confers antibiotic resistance to ampicillin.

Step 2: Treatment of plasmid and foreign DNA with the same restriction enzyme.

 The restriction enzyme XmaI cuts plasmid DNA at the restriction site.
 The foreign DNA is cut into one site by the same restriction enzyme;
 When the restriction enzyme cuts, it produces sticky ends on both the foreign
DNA fragments and the plasmid.

Step 3: Mixture of restriction digested foreign DNA and specific plasmid.

 Sticky ends of the plasmid will base pair with complementary sticky ends of
foreign DNA fragments.

Step 4: Addition of T4 DNA ligase.

 T4 DNA ligase catalyzes the formation of covalent bonds, joining the two
DNA molecules and forming a new plasmid with recombinant DNA.

Step 5: Introduction of recombinant plasmid into competent bacterial cells.

 T4 DNA is added to a competent bacterial culture.


 Some bacteria will take up the plasmid DNA by transformation.

Step 6: Production of multiple gene copies by gene cloning and selection process for
transformed cells.

8
 Bacteria transformed with the recombinant plasmid are allowed to reproduce,
cloning the inserted gene in the process.
 Recombinant plasmids can be identified by the fact that they are ampicillin
resistant and will grow in the presence of ampicillin.

Step 7: Final screening for transformed cells.

 Ampicillin is added to the culture medium to select plasmids


containing the foreign insert.

Objectives: Cloning of 1,8-cineole synthase gene into the recombinant


pQE10 vector.

9
2. Material and methods

2.1 Recombinant plasmid pQE-10

The recombinant plasmid pQE-10 with the mCherry sequence cloned was used in the
present study. Mcherry is a red monomer which matures extremely rapidly, making it possible to
see results very soon after activating transcription. Proteins with the tag mcherry, shows a pink
color. It is highly photostable and resistant to photobleaching.

Fig.1 pQE-10 with mCherry


sourse: http://www.snapgene.com/resources/plasmid_files/qiagen_vectors/pQE-10/

10
2.2 Transformation of DH5α E.colli cells

Transformation efficiency is defined as the number of colony forming units (cfu) which
would be produced by transforming 1 µg of plasmid DNA into a given volume of competent
cells. The term is somewhat misleading in that 1 µg of plasmid is rarely actually transformed.

Miniprep Protocol

1. Inoculate 5 ml LB medium (containing antibiotic like ampicilin 75μL/mL) with a bacterial


clone transformed with vector pQE10-mcherry and another one transformed with vector
pGEM T-Easy-1,8 cin. Both bacterial cultures growth overnight culture with vigorous
shaking at 37 degree for 14-16 hrs.
2. Take 1,5 mL of each bacterial culture in sample that containts the vector pQE10-mcherry and
1,5 mL that contains the pGEM T-Easy-1,8cin.
3. Centrifuge at 13000 rpm for 1 minute.
4. Discard the flow-through.
5. Add solution P1 ( 200 μL GTE + 10 μL RNAse), resuspend the cells and leave 10 minutes at
RT.
6. Add 200 μL solution P2 (NaOH 5M – 48 μL; SDS 1% - 120 μL; H2O – 1032 μL).
7. Leave 10 minutes at RT.
8. Add 200 μL solution P3 (NaAC 3M pH 5,5).
9. Put the samples in the ice for 30 minutes.
10. Centrifuge for 5 minutes at 13000 rpm 4°C.
11. Discard the flow-through and add 800 μL isopropanol.
12. Leave 5 minutes in ice.
13. Centrifuge for 15 minutes at 13000 rpm 4°C.
14. Discard the flow-through and wash with 1 mL ETOH 100% that stayed at -20°C.
15. Centrifuge for 15 minutes at 13000 rpm 4°C.
16. Discard the flow-through and wash with 1 mL ETOH 75% that stayed at -20°C.
17. Centrifuge for 15 minutes at 13000 rpm 4°C.
18. Discard the flow-through and wash with 1 mL ETOH 75% that stayed at -20°C.
19. Centrifuge for 5 minutes at 13000 rpm 4°C.
20. Discard the flow-through and leave for the samples to dry.
21. Add 30 μL H2O and put the samples at -20°C.

We will use 2 MiniPreps ( one of them is 1,8-cineole and the other Mcherry):

a) 1,8 cineol cut with enzyme XmaI;


b) Mcherry cut with enzyme XmaI

11
1. We will use 1μL of the competent cells and put into the MP’s.(under the flame so that
can’t be any contaminations)
2. The samples will stay in ice for 30 minutes.
3. For 1min and 30 second the samples will stay at 42°C (heat shock).
4. We cool down the samples for 2 minutes in ice.
5. Add 200μL medium LB in each samples.
6. Incubate for 30 minutes at 37°C.
7. Centrifugate for 2 minutes at 5000-6000 rpm.
8. Throw away the supernatant, and resuspend the pellets from the sample’s wall in 100μL
solution.
9. Spread the 100μL in Petri’s plates, that have LB medium with Ampicilin near the flame.
10. Incubate the plates for 16h at 37°C.

Digestion reaction of pQE10-mcherry with XmaI

Buffer 10x H20 DNA MP pQE10- XmaI


mcherry

1 μL 7,5 μL 1 μL 0,5μL

The 10 μL from the eppendorf sample will be put for 1h and 30 min at 37°C, and after
that we will make the electrophoresis and we will load the samples like in the table below. (fig.2)

Marker Digestion sample MP pQE10-mcherry MP 1,8cineol from


10.03.17

4 μL 10 μL 1 μL 1 μL

Fig.2 Loading Samples

For this experiment were made 3 digestions reactions using the same protocol.

12
2.3 Electrophoresis Gel

Pouring a Standard 1% Agarose Gel according to addgene protocol electrophoresis gel:


1. Measure 0,5 g of agarose.
Note: Agarose gels are commonly used in concentrations of 0.7% to 2% depending on the
size of bands needed to be separated. Simply adjust the mass of agarose in a given volume to
make gels of other agarose concentrations (e.g., 2 g of agarose in 100 mL of TAE will make a
2% gel).
2. Mix agarose powder with 50 mL 1xTAE in a microwavable flask.

3. Microwave for 1-3 min until the agarose is completely dissolved (but do not overboil the
solution, as some of the buffer will evaporate and thus alter the final percentage of
agarose in the gel).
4. Let agarose solution cool down.
5. Add GREENSAFE working solution (dil 1:10)
GreenSafe working solution (dil 1:10) Agarose Gel (mL)
10 μl 50 mL
30 μl 150 mL
35 μl 175 mL
40 μl 200 mL

6. Pour the agarose into a gel tray with the well comb in place.
Note: Pour slowly to avoid bubbles which will disrupt the gel. Any bubbles can be pushed
away from the well comb or towards the sides/edges of the gel with a pipette tip.
7. Let the gel sit at room temperature for 20-30 minutes, until it has completely solidified.

Loading Samples and Running an Agarose Gel:


1. Add loading buffer to each of your digested samples.
Note: Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel
loading and will also allows you to gauge how far the gel has run while you are running your
gel; and 2) it contains a high percentage of glycerol, so it increases the density of your DNA
sample causing it settle to the bottom of the gel well, instead of diffusing in the buffer.
2. Once solidified, place the agarose gel into the gel box (electrophoresis unit).

13
3. Fill gel box with 1xTAE until the gel is covered.
4. Carefully load your samples into the additional wells of the gel.
5. Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the
gel.
6. Turn OFF power, and then carefully remove the gel from the gel box.

Analyzing the Gel:

1. Using the DNA ladder in the first lane as a guide, you can interpret the bands that you get
in your sample lanes to determine if the resulting DNA bands that you see are as expected
or not.
2. Using any device that has UV light, visualize your DNA fragments.
Note: When using UV light, protect your skin by wearing safety goggles or a face shield,
gloves and a lab coat.
Note: If you will be purifying the DNA for later use, use long-wavelength UV and expose for
as little time as possible to minimize damage to the DNA.
Note: The fragments of DNA are usually referred to as ‘bands’ due to their appearance on
the gel.
After the process, we extract from the gel the recombinant plasmid pQE10 and the gene 1,8-
cineol.

14
Measuring the concentration of DNA through Nanodrop

For our samples we used the Thermo Nanodrop like in figure 3:

MP 1,8Cin XmaI col1 3971,5 ng/μL

MP 1,8Cin XmaI col2 4256,5 ng/μL

MP Mcherry col1 4319,7 ng/μL

MP Mcherry col2 2530 ng/μL

Fig.3 Thermo nanodrop

For the samples that was verified to have RNA, we add 3,5 μL of RNAse for 30 μL of
MP1,8Cin and 1μL for MP Mcherry PQE-10 col1, because the electrophoresis showed us that
this samples has a quantity of RNA.

15
2.4 Purification of gels from agarose of the MiniPreps using
Megaquick-spinTM Total Fragment DNA Purification Kit

For this method we used the protocol that is described in the kit purification.

The minipreps were weighted before we put the gel inside and after we put the gel. The result
between those two numbers was multiplied by 3, meaning the quantity of BNL Buffer that we
must add. For example if the sample has 0,200g we must add 600 μL of BNL Buffer.

Steps for the purification method:

1. After we put the BNL Buffer, the samples must be put into 75°C so that the gel
can be melted.
2. The solution from sampled will be put into Megaquick-colomnsTm.( only 800 μL
can fit into these colomns).
3. Centrifuge for 1 minute at 13000 rpm.
4. Discard the flow-through and wash with 700 μL Washing Buffer every sample.
5. Centrifuge for 1 minute at 13000 rpm.
6. Discard the flow-through.
7. Spin again so that the membrane will be dry.
8. The Megaquick-columns will be discarded intro new eppendorfs and add 30-40
μL H2O, leave for 1 minute at RT.
9. Centrifuge 1 minute at 13000 rpm.
10. Leave them at -20°C.

After the purification the samples were measured at the nanodrop:

MP Mcherry 345 ng/μL


MP 1,8Cin 805 ng/μL

Precipitation of DNA
We had 6 samples from MP pQE10-mcherry and MP 1,8Cin that need to be precipitated
by using this protocol:

1. Centrifuge for 15 minutes at 15000 rpm.


2. Discard the flow-through and add 1000 μL ETOH 70% that stayed at -20°C.
3. Centrifuge for 15 minutes at 15000 rpm.
4. Discard the flow-through and add 200 μL ETOH 100% that stayed at -20°C.

16
5. Centrifuge for 15 minutes at 15000 rpm.
6. Discard the flow-through and add 40 μL H20.

Measure all the samples at the spectrophotometer.

CIAP TREATMENT

First we make a Ciap Dilution that it will be used for CIAP TREATMENT.

CIAP Dilution:

H2O 10 μL
Buffer AP 10X 1 μL
CIAP 1U/μL + 10 μL buffer diluted 1:10 0,1 μL

CIAP TREATMENT:

MP pQE10-mcherry CIAP Buffer CIAP Dilution H2O


43,5 μL 5 μL 1,5 μL 0 μL

The sample will stay for 60 minutes at 35°C and at every 15 minutes agitate the sample.
After 30 minutes passed add 1,5 μL CIAP Dilution. After 1 hour add 300 μL CIAP Stop Buffer
and 200 μL fenol:chloroform:alcohol isoamilic (25:24:1).Spin for 2 minutes at 13000 rpm. All
the discard flow-through will be put into a new eppendorf and the volume will be checked. Add
320 μL of alcohol isoamilic. Spin for 3 minutes at 13000 rpm. All the discard flow-trough will
be put again into a new eppendorf and check the volume inside. After this step add 28 μL NaAC
pH 5,2 3M for MP1,8 Cin and 27 μL for MP Mcherry and 700 μL ETOH 100% for 1,8C and 675
μL for mcherry. Put the samples at -20°C.

A new precipitation of DNA was made on 23.06.2017 using the same protocol as the last
time. At the end of the method we did the ligation reaction.

Ligation Reaction

MP pQE10-mcherry CIAP XmaI 3 μL


MP 1,8Cin XmaI 0,5 μL
10x buffer T4-DNA ligase Thermoa Sci 1 μL
T4 DNA ligase (1 wess unit) 0,2 μL
H2O 5,3 μL

17
We let the sample stay for the weekend at 4°C.

Transformation of DH5α competent cells with the ligation

The ligation has to be inactive when we insert into competent cells, so will put the
ligasion for 5 minutes at 80°C.

1. Add 5μL of ligasion into DH5α competent cells, under the flame.
2. Leave the sample 30 minutes on ice.
3. For 1min and 30 second the samples will stay at 42°C (heat shock).
4. We cool down the samples for 2 minutes in ice.
5. Add 400μL medium LB in each samples.
6. Incubate for 1 hour at 37°C.
7. Centrifuge at 5000 rpm for 2 minutes and discard about 100μL solution into a
new eppendorf, and resuspend the cells.
8. Put the solution into the plates and spread it and leave for incubate for 16h.

A new ligation was made on 22.06.17 using MP Mcherry CIAP XmaI and MP1,8Cin
XmaI following the same protocol as in the last ligation.

The ligation stayed at 4°C for the weekend and after that a new transformation was made
using the same protocol.

The next stept was using a PCR protocol following the table below:

Mix 15 μL x3
Buffer VWR 10X 1,5 4,5
MgCl 25mM 0,9 2,7
dNTP’s 10 mM 0,3 0,9
Primer FW 49 10 μM 0,3 0,9
Primer RV 48 10 μM 0,3 0,9
Taq VWR 0,1125 0,338
H20 11,587 19,76

5 μL of H2O were add to each colony and it was put at 98°C for 4 minutes.

For x35 cycles we used next temperatures:

97°C 2 minutes
94°C 30 seconds
58°C 40 seconds

18
72°C 1,40 minutes
72°C 5 minutes

After the PCR is done we made with the final products an electrophoresis using:

Marker MP1,8CIN C+ C-
4 μL 5 μL 5 μL 5 μL

On 4.07.2017 we tested the 2nd Mcherry colony by doing another PCR. Cut the Mcherry
with XmaI and transformation of ligation with competent cells.

For the PCR we used the same protocol, followed by the electrophoresis.

The transformartion was done using DH5α and JM107 compentent cells with MP1,8Cin
from 31.10.15 using the protocol for transformation cells. Results: no colonies.

After this a new digestion reaction was made with MP Mcherry col2 from 16.2.17 and
with MP1,8 Cin from 13.6.17. The results were verified through electrophoresis.

The gel was cut and put into two samples: MP mcherry and MP 1,8Cin. The following
step was to purify those sample using the same protocol, and that is Megaquick-ColomnTM
Total DNA Fragment Purification Kit. After the purification, the probes were measured using
nanodrop. Using this we can assure that we can do the following steps and that is CIAP
TRATAMENT. The Ciap tratament is the same as it has been explained before. At the end of the
tratament the samples will stay at -20°C.

The next method concerned the transformation of the ligtaion made on 27.06.17 with
JM107 competent cells by inactive this ligation at 78°C for 5 minutes and following the same
protocol for transformation, and also the transformation of MP Mcheery from 5.12.12 with the
same competent cells JM107. After the last step, referring to spreading on the plates, they will be
staying for 16h on incubating.

The results was that there were no colonies.

Precipitation of DNA with sample from 05.07.17

Using the same protocol of precipitation the sample was verified through electrophoresis.

On 06.07.2017 a new ligation was made using this table:

19
10x Buffer T4 DNAse ligase Therma Sci 1 μL
T4 DNAse ligase (5 weiss/unit) 0,4 μL
H2O 5,1 μL
MP mcherry Ciap XmaI 5.7.17 1,5 μL
MP 1,8Cin Purify 5.7.17 2,5 μL
Leave the sample for 20 minutes at RT and at 4°C for the weekend.

With this ligation we made another transformation with DH5α competent cells on
10.7.17, but the next day they were not results on the plates.

Preparing the competent cells

Steps in making competent cells:

1. 100 μL bacterian solution that was rised ON diluted with 10 mL LB.


2. Incubate for 2h at 37°C at 200 rpm.
3. Spin for 10 minutes at 3000 rpm 4°C.
4. Discard the flow-through and add 10 mL CaCl 0,1M at 4°C.
5. Leave the sample 1h on ice.
6. Spin for 3000 rpm for 10 minutes at 4°C.
7. Discard the flow-through and add 1 mL CaCl 0,1M at 4°C.
8. Resuspend the cells and put 50 μL or 100 μL per each transformation you need.
Transformation of ligation from 05.07.17 with the new competent cells

We used 3 μL of ligation and put them into a sample that has new compentent cells made on
11.07.17. The steps are the same as in every transformation protocol. At the final of
transformation we spread the culture in plates and leave them from incubation 16h.

There were results on these plates and we made a PCR using 3 colonies. The method for PCR
was the same as in protocol that I have written above.

Following the PCR was the electrophoresis.

The colonies that have growth we put them into tubes for growing more for 16h at 37°C shaker
200rpm and the plate was put into incubator also at 37°C.

20
Making the MiniPreps with PUC57-18-CE-GFP and Mcherry

We put for both sample 1,5 mL solution. The samples were centrifugate for 1 min at
13000 rpm. The next steps are the same as in the previous protocol, making solution P1, P2 and
P3 and washing the samples with ETOH 75% that stayed at -20°C.

After those MP’s were done we made a PCR using 1,5 μL of all 4 samples( PUC-18-
CE1, PUC-18CE2, Mcherry1, Mcherry2).

With the final product of the PCR we made an electrophoresis and we load the sample
using this table:

Marker Product1 Product 3 C+ C-


4 μL 5 μL 5 μL 5 μL 5 μL

DNA Clean Up Purification

With the samples from 13.7.17 MP PQE-10 1,8Cin we made a purification, using 25 μL
solution in an eppendorf, following the steps from the protocol:

1. Add 100 μL BNL Buffer.


2. Centrifuge for 1 minute at 13000 rpm.
3. Put the solution in a Megaquick-Colomn.
4. Centrifuge for 1 minute at 13000 rpm.
5. Discard the flow-through and add 700 μL Washing Buffer.
6. Centrifuge for 1 minute at 13000 rpm.
7. The Megaquick-Colomn is transported into another eppendorf.
8. Add 30 μL Elution Buffer.
9. Stays for 2 minutes at RT.
10. Centrifuge for 1 minute at 13000 rpm.
11. Put the samples at -20°C.

21
pCAMBIA-CTV vector with PUC57-18-CE-GFP

For date 18.07.17 we had to make a transformation with DH5α competent cells with
ligation from 5.7.17, a digestion reaction using PUC57-18-CE GFP with PstI, and another
digestion reaction using pCAMBIA-CTV vector with PstI/Fast AP.

Digestion of PUC57-1,8CE GFP with PstI:

10x Buffer H 3 μL
PstI 1 μL
H2O 21 μL
MP PUC57-18-CE-GFP 5 μL
We put the sample for 2h at 37°C

Digestion of pCAMBIA with PstI:

10x Buffer H 3 μL
PstI 0,8 μL
H2O 21,2 μL
MP pCAMBIA 5 μL
We put the sample for 2h at 37°C, but after 1h and 40 minute we added Alkaline
Phosphotase to the sample.

After the incubation the samples were verified through electrophoresis:

Marker Dig pCAMBIA Dig pCAMBIA Dig PUC57-18- Dig PUC57-18-


CE GFP CE GFP
4 μL 15 μL 15 μL 15 μL 15 μL

Making the MiniPreps on 19.7.17 using Mcherry that was growth over night

We put into 2 samples 1,5 mL of solution and centrifugate for 1 minute at 13000 rpm.
Discard the flow-through and like in the others protocols that were written above we used the
same solutions P1, P2, P3, and washed the samples with ETOH 100% and ETOH 75%. After we
let the samples to dry of ETOH we add in one sample 30 μL H2O and in the other sample 30μL
Elution Buffer and then we can make an electrophoresis gel and verify them.

22
Following the electrophoresis we did a purification for the samples, using the protocol for
Megaquick-ColomnSpinTM Total DNA Fragment Purification Kit. After the purification we
made another electrophoresis like in the table bellow and in the figure 5

Marker MP Mcherry 1,8C


3 μL 1 μL
and after that we put the samples at -20°C.

M MP

Fig.5 Electrophoresis of the loading


samples

3. Results

As for the results we can say that we were succeded in our objectives: to amplify the gene
1,8-cineole and clone into the recombinant expression vector pQE-10- mcherry. The next step
will be the heterologous expression of the protein 1,8-cineole.

23
4. Bibliography

1. Alberts B, Bray D, Lewis J, Raff M, Roberts K (1994) Molecular biology of the


cell. Garland Publishing, New York.
2. https://www.ncbi.nlm.nih.gov/probe/docs/techpcr/
3. http://www.snapgene.com/resources/plasmid_files/qiagen_vectors/pQE-10/
4. http://www.clontech.com/US/Products/Fluorescent_Proteins_and_Reporters/Fluor
escent_Proteins_by_Name/mCherry_Fluorescent_Protein
5. https://www.ncbi.nlm.nih.gov/books/NBK21450/
6. https://www.neb.com/tools-and-resources/usage-guidelines/chemical-
transformation-tips

24