Dear Jo Erickson,

due to your problem with the cell line recommendations for transfection of plasmiid vektor with human insert, I am making this more simple with the adding of the Stndard protocol for cloning of GFP into Mammalian cell line. So, on this basis you can make transfection of your plasmid. I wish you a good luck!

If you need more, please contact me.

Sincerely yours,

Dr. Bratko Filipic

Email: [email protected]

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Protocol for Cloning GFP into Mammalian cells

Content Introduction....................................................................................................................................................... 2

Day 1.................................................................................................................................................................. 4

Plasmid production: Ligation of the GFP gene into an expression plasmid.................................................. 4

Materials.................................................................................................................................................... 4

Protocol ..................................................................................................................................................... 4

Cell Transfection: The GFP plasmid is transfected into mammalian cells..................................................... 5

Materials.................................................................................................................................................... 5

Protocol ..................................................................................................................................................... 5

Day 2:................................................................................................................................................................. 6

Plasmid production: Transformation of E. coli cells with ligation mixes....................................................... 6

Materials.................................................................................................................................................... 6

Protocol ..................................................................................................................................................... 6

Cell transfection: Maintenance of cells......................................................................................................... 7

Materials.................................................................................................................................................... 7

Protocol ..................................................................................................................................................... 7

Day 3.................................................................................................................................................................. 8

Plasmid production: Check colonies for inserted GFP by colony PCR........................................................... 8

Materials.................................................................................................................................................... 8

Protocol ..................................................................................................................................................... 8

Cell transformation:GFP-glowing cells ........................................................................................................ 10

Materials.................................................................................................................................................. 10

Protocol ................................................................................................................................................... 10

Appendix.......................................................................................................................................................... 11

Map of pEGFP-N1........................................................................................................................................ 11

The sequence of pEGFP-N1......................................................................................................................... 12

100 bp DNA size marker.............................................................................................................................. 15

How to load an agarose gel......................................................................................................................... 16

1

Introduction

In your three days at Aarhus University you are going to conduct an experiment in order to make human cells emit green fluorescent light. This is done by using the gene of green fluorescent protein (GFP) from jellyfish. We have prepared the gene for you to work with, consisting of a double stranded piece of DNA. You will also receive a flask of living mammalian cells. In order to get the gene into the cells, you have to convert the gene into a circle of DNA, a so called plasmid. Plasmids are more easily taken up by cells, than linear pieces of DNA, and do not have to be inserted into the genome of the host, but will functions as an individual small genome alone.

To prepare the plasmid, you will receive an already existing ring of DNA, the expression plasmid, which we have cut open for you. The GFP gene is ligated into the open plasmid to recreate a circle. To ligate, an enzyme called T4 ligase is used. The outcome is a closed plasmid containing our GFP gene.

In the next step we want to sort and amplify our newly prepared GFP plasmid. Our reaction mixture from above contains both unclosed and closed circles of DNA, and some maybe with a wrong insert. To sort out the plasmids with a flawless GFP insert, we will begin with transforming all plasmids from the sample into E. coli bacteria cells. E. coli easily takes up plasmids, and once taken up, the cell will believe the DNA to be part of its genome, and will thus pass on a copy of the plasmid to future daughter cells. This means that one single E. coli cell, which has taken up the GFP plasmid, will give rise to a whole colony of bacteria cells all expressing the plasmid harboring our GFP gene. To get a sufficient amount of plasmid, we also have to rely on E. coli cell to mass produce our GFP containing plasmid.

To select the single E. coli cells which have taken up the GFP plasmid, and to transform this cell into a small colony, we seed the cells from above on media plates. Our GFP plasmid contains, together with GFP, a gene for the antibiotic kanamycin resistance. This means that E. coli cells which have taken up our plasmid, also are resistant to kanamycin. Cells which have not taken up any plasmid will die in the presents of kanamycin. So to sort out the cells, we grow them on plates containing kanamycin.

E. coli colonies surviving on the plates have the resistance gene, but we have to test whether they also contains the GFP gene. A plasmid closed without GFP insert, still has resistance towards kanamycin. This is done with PCR, where DNA can be amplified and visualized through gel

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electrophoresis. We test for GFP and will in our gel see which E. coli colonies contains the real gene.

The next step we will perform for you, since we have no time for it together. The colonies positive for GFP will be transferred to a flask with media to grow. When a sufficient number of cells are reached, they are harvested and lysed (broken open) and the plasmids are purified.

Now you have your plasmid containing the GFP gene and you are ready to put it into mammalian cells. This is done by using calcium phosphate transfection. That transfection method is based on forming a calcium phosphate-DNA precipitate, which facilitates the binding of the DNA to the cell surface. DNA then enters the cell by endocytosis. This means that the cell membrane, which covers the cell, will fold around the DNA and drag it into the cell.

After transfection, the cells are allowed to grow for two days. On the second day you will have to change their media in order for them to maintain healthy. On the third day the GFP protein have been expressed in appropriate amounts to be visualized under UV light. This means that you can see your cells glowing in a green light if you look in a microscope.

📷The procedure above takes six days of work, and since you only have three, we will divide the

experiment in two. This means that you on day one will begin by putting GFP into a plasmid as explained. At the same day, you will receive some already made GFP plasmid which you will transfect into mammalian cells. On your last day, you will thus hopefully have an E. coli colony expressing plasmid containing GFP, and mammalian cells green from GFP protein.

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Day 1

Plasmidproduction: Ligation of theGFP geneintoan expression plasmid You are going to ligate GFP into an already cut open expression plasmid. By ligation the GFP

becomes a covalent part of the plasmid which is at the same time circularized. It is only the circular form that can be replicated inside cells.

The GFP and the plasmid have both been cut with the restriction enzymes SalI and NotI, which recognizes specific DNA sequences. This means that the gene and the plasmid have ends matching each other. A map and sequence of the plasmid with GFP (called pEGFP-N1) inserted is in the appendix.

Materials

The purified restricted GFP

10 x T4 Ligase buffer T4 DNA Ligase Water

Restricted plasmid

Protocol

1. Prepare the following ligation mixes for your restricted GFP: (amounts in µL)

Sample A B

Water 16 11 10 x T4 Ligase buffer 2 2 Restricted GFP 0 5 Restricted plasmid 1 1 T4 DNA Ligase 1 1

2. Incubate the ligation mixtures at 16°C in a heat block until next day.

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CellTransfection:The GFP plasmid is transfected into mammalian cells To get the new GFP plasmid into the mammalian cells, the plasmid is mixed directly with a

concentrated solution of CaCl2. This mixture is then added drop wise to a phosphate buffer to form a fine precipitate. Aeration of the phosphate buffer while adding the DNA-CaCl2 solution helps to ensure that the precipitate that forms is as fine as possible, which is important because clumped DNA will not adhere to or enter the cell as efficiently. This solution is then added drop wise to the cells.

The cells used in this experiment are a line of immortalized human cells. They grow on an active surface on the bottom of a plastic bottle. Be careful not to disturb the cells. They have to stay attached to the bottom of the bottle in order to remain healthy.

Materials Human cells

2.5M CaCl2 Plasmid Hepes buffer

Protocol

1. Add 50 µl 2.5M CaCl2 to your tube containing 450 µl DNA. Mix by taking the liquid up and

down with your pipet. Avoid air bubbles.

2. Add the 500 µl DNA-CaCl2-solution slowly one drop at a time to the tube containing 500 µl 2x Hepes buffer. While you do this, you continuously make bubbles in the solution with a bigger pipet.

3. Leave the mixture for 5 min at room temperature.

4. Add the mixture to your cells one drop at a time.

5. Leave the cells to grow in the incubator overnight.

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Day 2:

Plasmidproduction: Transformation of E. coli cells with ligation mixes

The plasmids carrying insert (GFP) are to be transferred into E. coli in order to be sorted and

replicated. This process is called transformation. The E. coli cells have been treated in such a manner that they are able to take up DNA. The cells are incubated with DNA plasmids and will, after heat shock at 42°C for 20 sec., take up the plasmids having accumulated on their cell surface. The transformed cells are plated on agar plates containing a selective antibiotic (here kanamycin). E. coli cells that have received intact plasmids will then be able to divide and grow into colonies on the agar plates, because the plasmids carry the gene for antibiotic resistance. Note, that the antibiotic resistance does not give any information whether the cells also have received the GFP gene.

Materials LB- medium

2 LB-agar plates containing kanamycin 2 Eppendorf tubes with E. coli cells. The ligation mixtures from yesterday

Plastic Drigalsky spatulas for plating the bacteria 42°C heat block

Protocol

1. Transfer 5 µL of each ligation mix (A and B) into separate tubes with E. coli. Use a pipet tip to

stir around.

2. Incubate the transformation tubes on ice for 30 min.

3. Heat-chok the cells in a 42°C heat block for 20 sec. and immediately thereafter incubate on ice for at least 2 min.

4. Add 950 µL LB-medium to each of the two transformation tubes.

5. Incubate at 37°C for 1 hour.

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6. Plate 150 µL of each transformation mix on each of two LB-agar plates marked A and B. Write

name and group on the plates.

7. The agar plates are incubated (bottom up!) in a 37°C incubator overnight.

Cell transfection: Maintenance of cells

Today the cells have to have their old media taken away, get washed and receive new media. The

media contains among other the nutrients the cells need to grow.

Materials Waste tube

PBS wash buffer Media

Protocol

1. Remove the old media from the cells, by transferring it to a waste tube.

2. Wash the cells with 5 mL wash buffer (PBS). Be careful not to disturb the cells. Add the buffer,

let it flow around and empty the flask again by transferring the buffer to the waste tube.

3. Add 10 mL of new media.

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Day 3

Plasmidproduction: Check colonies for insertedGFP by colony PCR

Today you are going to find out whether any of your E. coli colonies contain the GFP insert.

You will be doing this by colony-PCR using a primer set where one primer anneals upstream of (before) the GFP gene and one anneals inside the gene itself. A PCR product of the right size tells you that the insert is most probably GFP and that it is oriented correctly in the plasmid.

Materials

You will get 55 µL of following stock mix made by the instructors:

5.5 µL 10 x SuperTaq buffer

0.55 µL SuperTaq DNA polymerase

2.2 µL CMV forward primer, 5 pmol/µL 2.2 µL eGFP – N reverse primer, 5 pmol/µL 1.1 µL 10 mM dNTP

43.45 µL water Eppendorf tubes

1% agarose gel (incl. GelRed) Gel apparatus

Power supply

6 x Ficoll DNA loading buffer (Ficoll pulls the dye down in the wells)

100 bp DNA size marker (see appendix for the size of each band in the marker)

Protocol

1. Number 5 PCR tubes (1-5).

2. Add 10 µL stock mix to each of the PCR tubes.

3. Mark the colonies that you want to test on the bottom of the plate with a pen.

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4. Pick the 5 colonies with a small pipette tip (those for Pipetman P10) by dipping the pipette tip into a colony and then dipping it in one of the PCR tubes and stir a bit about, so that the cells gets into the liquid. Close the lids on the PCR tubes.

6. The 5 PCR tubes are placed in the PCR machine (remember team numbers!) and the PCR is started.

7. The PCR cycle program is:

1. Initial denaturation of template DNA:

2. Amplification cycles (repeated 30 times):

2 min at 95°C

30 sec. at 95°C (melting) 30 sec. at 60°C (annealing)

1 min. at 72°C (elongation)

8. After ended PCR add 2 µL 6 x Ficoll DNA loading buffer to each PCR tube and to the size

marker.

9. Load the agarose gel with the entire volumes of each sample. Write down where you load the samples. (See appendix for how to load a gel).

10. Run electrophoresis at 60 mA until the blue dye is around 2.5 cm from the bottom of the gel.

11. Visualize the agarose gel on a UV box and photograph your gel.

12. Write up sample numbers on your photograph.

- Do all the samples contain the plasmid with the insert?

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Cell transformation: GFP-glowing cells

Today you will see whether the transformation has worked.

Materials

Fluorescence microscope

Protocol

1. Look at your cells in a microscope.

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agree with James Gilbert

I also highly recommend HEK cells. I've been using these cells for transfection since 2017 and it's been great. The maintenance is easy. They don't grow very fast or very slow. They also are very good with different transfection reagents.