4BBY1070 Practical 1 guidance PDF

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Practical 1. Restriction enzyme digestion of plasmid pGLO and analysis using agarose gel electrophoresis. Learning outcomes After completing the practical and the associated proforma students should: 1. Be aware that agarose gel electrophoresis is used to analyse the size of DNA fragments. 2. Be able to construct a standard curve using DNA fragments of known size. 3. Be able to use the data obtained to confirm the identity of a plasmid. Introduction Type II restriction enzymes (restriction endonucleases) cut double stranded DNA molecules at specific sites defined by the DNA sequence. After the DNA molecule has been digested by restriction enzymes, the DNA fragments produced can be separated by agarose gel electrophoresis. In this practical you will digest plasmid pGLO (shown in figure 1) with three restriction enzymes, EcoRV, HindIII and PstI which recognise different sites in plasmids DNA sequence. The digested plasmid DNA will be run on an agarose gel to analyse the size of the DNA fragments produced. The sizes of the fragments of DNA will be determined by comparison with DNA marker fragments of known size (bp) and the data used to check the identity of plasmid pGLO. Additional information for Practicals 1 and 2 can be found on the KEATS site in document called Background information for 4BBY1070 Practicals 1 and 2.

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Figure 1. pGLO plasmid map showing the position of AmpR, araC and GFP genes, the araBAD promoter, the origin of replication, and position of EcoRV, BamHI, HindII, PstI , XhoI and XbaI restriction enzyme sites.

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Materials (each group of two students) 2x 4BBY1070 Practical Handbooks 1x permanent black marker pen 1x ice bucket with ice 1x 1.5 ml tube rack 1x 20 µl Gilson pipette 1x box of sterile pipette tips 1x box sterile 1.5 ml tubes 1x 200 µl sterile dH2O in a 1.5 ml tube labelled H2O on the lid 1x 20 µl FastDigest Buffer in a 1.5 ml tube labelled 10xFAST on the lid 1x 70 µl pGLO (10ng/µl) in a 1.5 ml tube labelled pGLO on the lid 1x 5 µl Fastdigest EcoRV in a 1.5 ml tube labelled EcoRV on the lid (on ice) 1x 5 µl Fastdigest HindIII in a 1.5 ml tube labelled HindIII on the lid (on ice) 1x 5 µl Fastdigest PstI in a 1.5 ml tube labelled PstI on the lid (on ice) 1x 30 µl Promega 6x Blue/Orange loading dye in a 1.5 ml tube labelled 6XBO dye on the lid (on ice) 1x 250 ml conical flask 1x 50 ml measuring cylinder 1x 250 ml measuring cylinder 1x Mini horizontal gel electrophoresis apparatus with gel tray and comb 1x sandwich box (big enough to contain the gel tray carrying the agarose gel). On bench 2x yellow cinbins 2x powerpacks 2x microfuge 1.5 ml tube rack with 1x 100 µl GelRed in a 1.5 ml tube labelled GelRed on the lid 2 x power packs (shared between two groups) 1x blue paper towel roll 1x stapler with staples 1x weighing scale 4x Medium weighting boats (one for each pair) 1x pot of agarose 1x spatula 1x microwave 4x pairs of heat resistant gloves (on top of microwave) In the lab Computer with internet access and AV and screens around class switched on before students enter and tie microphone with spare battery nitrile gloves (selection sizes) safety glasses 2 x 10 litre Dewars containing 1xTAE 2x image analysers with printers with paper 1x roll autoclave tape (to mark gel position on UV light box) 5x 37°C heating blocks with 1.5 ml tube metal inserts (enough for 5 holes per pair students)

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Part 1. Preparation of a 1% w/v 1xTAE agarose gel a. Measure 50 ml of 1x TAE in a 50 ml measuring cylinder and place this in a 250 ml conical flask. Weigh out 0.5g of agarose into a weighing boat. Add the 0.5g of agarose into the 50 ml of 1xTAE in your conical flask 1xTAE (try not to get solid agarose stuck to the sides of the conical flask as it will not melt into solution easily if you do this). b. Heat in microwave at full power for 1 – 2 minutes until solution has just started to boil (watch liquid in flask through window). Take the flask out of microwave wearing heat resistant gloves and swirl gently while another student is microwaving their flask. Now reheat your agarose solution for about 15 seconds in the microwave until liquid is just starting to boil again. This ‘double boil’ method works well and ensures all the agarose has melted into the 1xTAE (there should be very few visible tiny lumps). NOTE. Do not over boil your agarose gel because the water will evaporate and increase the agarose concentration and make it harder for the larger DNA fragments to separate into distinct ‘bands’). Bring the gel mixture to the point it is just starting to boil in the microwave. c. Add 5 µl of GelRed to the melted agarose and swirl. d. Remove the lid from the gel tank by using the white poles and the edge of the lid as leavers. Do NOT take the lid off by pulling the red and black wires/connectors as this can damage the electrical connections. First make sure that the two rubber seals are firmly in the groves at the edges of the gel tray and they are not stretched. Turn the tray so the edges are sealed by the sides of the gel tank. After the agarose has cooled for few minutes pour it into the gel tray. Place one 8 well comb in the slot at one end of the tray (with wells facing furthest from edge of the tank). Allow the agarose gel to set. You will need to leave the agarose gel to set for at least 30 minutes before you remove the comb. Record the time you poured your agarose gel here......................... e. At the nearest sink, rinse your conical flask out with water from the tap and return the rinsed flask to your bench. f. Throw your used weighing boat in the nearest clinical waste bin (the ones with yellow plastic bags).

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Part 2. Setting up the restriction enzyme digests of plasmid pGLO a. Spin the six tubes listed below in the microfuge to make sure all the liquid is at the bottom of the tubes (a 5 second pulse is enough). As with all centrifuges the rotor must be balanced with tubes of equal weight on opposite sides of the rotor head. Failure to balance a rotor will damage the centrifuge. Ask for help to balance and use your microfuge if you are unsure. EcoRV HindIII PstI pGLO 10xFAST 6XBO dye b. Label five new 1.5 ml tubes 1 to 5 on both the lids and the sides of the tube with the black marker pen. Pipette the following volumes at the bottom of each tube using a 20 µl Gilson pipette and sterile tips. You must use a new tip for each addition. Check to make sure you have taken up and dispensed these small volumes by looking at the tip before and after you have placed each component in the appropriate tube. You should add the water (H2O) to all five tubes first. Then the 10xFAST to the five tubes next and so on down the table. Tick each addition off on the table below as you add it to the tube.

Tube 1

Tube 2

Tube 3

Tube 4

Tube 5

H2O

8 µl

7 µl

7 µl

7 µl

6 µl

10xFAST

2 µl

2 µl

2 µl

2 µl

2 µl

pGLO

10 µl

10 µl

10 µl

10 µl

10 µl

EcoRV

none

1 µl

none

none

1 µl

HindIII

none

none

1 µl

none

none

PstI

none

none

none

1 µl

1 µl

TOTAL volume

20 µl

20 µl

20 µl

20 µl

20 µl

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c. Mix the contents of the tubes by setting your Gilson pipette to 18 µl and pipetting the mixture up and down two times using a new tip for each tube. Spin the five tubes in a microfuge for 10 seconds to make sure all the contents are at the bottom of the tube. d. Write your initials on the lids so you can identify your tubes. Put your five tubes in a 37°C heating block for at least 15 minutes. The time I put the five tubes at 37°C is ............................... Note. You can leave your five restriction digest tubes in the 37°C heating block until the agarose gel has been allowed to set for 30 minutes. In this type of experiment a bit more time ‘digesting’ does not harm the results.

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Part 3. Loading and running the pGLO restriction enzyme digests on a 1% w/v 1xTAE agarose gel a. After at least 15 minutes incubation remove your five restriction digest tubes from the 37°C heating block and place in the rack. b. Add 4 µl of 6xBO dye to each of your five tubes using a new tip each time. NOTE. 6xBO dye is a DNA loading buffer containing coloured dyes and a density agent called Ficoll (a soluble high molecular weight polysaccharide). The density agent increases the density of the DNA sample allowing the sample to sink into the bottom of the well. c. The agarose gel will take 30 minutes to set fully. When the gel has set you need to turn the gel tray so that the open ends of the gel are facing the electrodes at each end of the gel tank. The end with the wells should be at the end of the tank that attaches to the black electrode (if in doubt line up the lid on the tank with the electrodes and work it out). NOTE. When you are moving your gel tray make sure you keep the tray horizontal –if you tip the tray your gel can easily slide off and breakinto pieces. d. Gently remove the comb making sure you lift it directly upwards with no side to side movement. e. Once you have positioned your gel in the tank pour in 1xTAE electrophoresis buffer (approximately 250 mls) to submerge the agarose gel. N.B. The buffer should be 3-4 mm above the surface of the agarose gel. f. The wells should be furthest from you when you load the gel in the tank (so effectively the gel is orientated with the wells at the top as you would present the image/photo in any scientific publication). You can now load 10 µl of the 1kb plus marker and 20 µl of each of the five digests into separate wells following the table below. Lane 1

Lane 2

Lane 3

Lane 4

Lane 5

Lane 6

1kb plus markers

Tube 1

Tube 2

Tube 3

Tube 4

Tube 5

10 µl

20 µl

20 µl

20 µl

20 µl

20 µl

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g. Place the lid on the gel connecting the negative electrode (black) to the end near the sample wells as the DNA molecules will migrate from the negative towards the positive electrode. The positive (red) electrode is connected at the other end of the gel. Switch the power pack on and set at a constant voltage of 120V (and check the current is set at maximum 400 mA). Look at the electrodes at each end of the tank – more bubbles at the black end indicate that the circuit is complete (the current is running) and you have connected the tank up correctly. The gel needs to be turned off when the orange dye band has just about completely run off the end of the gel (this should take about 40 minutes from the time the current was switched on but the exact time will vary from gel to gel). NOTE. The Promega 6x Blue/Orange Loading Dye (6xBO dye) used in this practical contains Ficoll and three different dyes: Xylene Cyanol FF (turquoise blue) that migrates on a 1 % w/v agarose gel at approximately 4000 bp, Bromophenol Blue (dark blue) that migrates at approximately 300 bp and Orange G (orange) that migrates at approximately 50 bp.

Figure 2. 1 kb plus DNA ladder (Invitrogen). The DNA fragments are linear double stranded DNA. https://www.thermofisher.com/order/catalog/product/10787018?SID=srch-srp-10787018

NOTE. The 1.5 kb (1500 bp) DNA fragment of this DNA marker should appear brighter (known as the reference band). You are unlikely to resolve the 7 to 15 kb DNA marker fragments on a 0.8% 1xTAE agarose min gel and you may have run the smaller marker DNA fragments (100 to 300 bp) off the end of the gel, so the 1.5 kb reference band is a useful starting point when you identify the sizes of the DNA marker fragments on your gel photograph.

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Part 4. Photographing the agarose gel The gel must be turned off when the orange dye has just about to run off the end of the gel. a. Wearing nitrile gloves carefully lift your gel tray with the gel into the sandwich box taking care to keep the tray horizontal as the gel can slip off the tray and break. Take your sandwich box containing the tray and gel to the demonstrator near the image analyser and he/she will photograph of your gel and give you a copy of the photo. b. Look carefully at the gel photograph. i. The wells should be visible on the gel photograph. ii. The 1 kb plus DNA marker bands should be visible on the gel photograph down to at least 5 kb. iii. The pGLO DNA fragments should be visible on the gel photograph. c. Staple your gel photo in the space provided below to keep it safe.

STAPLE YOUR GEL PHOTO HERE BEFORE YOU LEAVE THE PRACTICAL CLASS SO YOU DO NOT LOSE IT

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