Restriction Digest and agarose gel electrophoresis

Overview:

Two procedures will be done during this lab period:

(1) Each team will set up their restriction digestion reactions, using the enzyme(s) that they chose;

(2) After the reactions are terminated, they will be run on agarose gels, along with the previously generated PCR products, for visualization of bands.

To make most efficient use of our time, please begin with the restriction digest set-up, and get those reactions incubating. While they are incubating, you can prepare aliquots of your PCR reactions for electrophoresis, and have them ready. Agarose gels have already been cast, and all you have to do is prepare your samples for electrophoresis and start the gels. Instructor will demonstrate.

Please note: if minor changes need to be made to the following information, this protocol will be updated and I will inform you.

General comments for restriction digestion: (specific reaction conditions follow):

Restriction digestion reaction set-up:

Total reaction volume:

For XcmI and SexAI: 50 ul
For StuI, XmnI, and DraIII: 20 ul

For each DNA sample, there will be a ‘cut’ sample and an ‘uncut’ control sample*.

* “uncut” = a control, run in parallel, without restriction enzymes added; “cut” refers to the reaction with restriction enzymes. Think about why we would need to include an ‘uncut’ control for each sample.

So, each team (2 plasmid samples) will have a total of 4 restriction digest reactions to set up (in 4 new eppendorf tubes):

DNA #1 (uncut) DNA #1 (cut)
DNA #2 (uncut) DNA #2 (cut)

The uncut controls include all reaction components in the tube minus enzyme(s); in those reactions, the corresponding volume of dH2O will replace the volume of enzyme(s) you would have used.

Each team is responsible for figuring out the expected sizes of their digested DNA fragments under their particular digest conditions. (NEB website provides this information.)

Note that the required incubation times are not identical for each enzyme; some enzymes (eg, XmnI, DraIII, StuI) can digest the DNA adequately in only ~15 min at 370C (although it won’t hurt the reactions to incubate longer). Other enzymes (eg, XcmI and SexAI) require the full 60 min (370C). Again, slightly longer incubation times should not hurt, so the timing is not critical here, so long as they are given the minimum time required.

  • Keep enzymes on ice! (The reaction tubes themselves, during set up, don’t have to be on ice)
  • You must calculate the volume of DNA (in ul) needed to provide 400ng- 1000ng (1 ug) per reaction tube; this volume will depend on your DNA concentrations. The required volumes of enzyme and buffer in each reaction tube are given below. Also, you will need to calculate the volume of dH2O required to bring each reaction to the total volume indicated (20 ul or 50 ul, depending on the enzyme). Remember to substitute dH2O for enzyme in the ‘uncut’ controls.
  • After the reaction tubes are set up, and before beginning incubation (see below for conditions), vortex (or ‘flick’) your digest samples briefly to mix, and then pulse-spin to bring contents to bottom of tubes; make sure tubes are tightly capped. After incubation is complete, pulse-spin samples to bring down condensation.

Then terminate each digest reaction by adding the indicated volume of 6X DNA loading dye (for the gels) directly to samples; then vortex and pulse spin again, to thoroughly mix:

-For XcmI and SexAI (50 ul reactions): add 10 ul of 6X DNA loading dye

-For StuI, XmnI, and DraIII (20 ul reactions): add 4 ul of 6X DNA loading dye

Proceed to gel.

Specific reaction conditions for each enzyme:

XcmI

Restriction enzyme                         2 ul
DNA                                                   0.4-1 ug
10X NEB buffer (“CutSmart”)        5 ul (for 1X final)
dH2O                                                  to 50 ul

Incubate at 370C, 60 min

SexAI

Restriction enzyme                         2 ul
DNA                                                   0.4-1 ug
10X NEB buffer (“CutSmart”)        5 ul (for 1X final)
dH2O                                                  to 50 ul

Incubate 370C, 60 min

XmnI

Restriction enzyme                         1 ul
DNA                                                   0.4-1 ug
10X NEB buffer (“CutSmart”)        2 ul (for 1X final)
dH2O                                                  to 20 ul

Incubate 370C, 15 min

DraIII

Restriction enzyme                         1 ul
DNA                                                   0.4-1 ug
10X NEB buffer (“CutSmart”)        2 ul (for 1X final)
dH2O                                                  to 20 ul

Incubate 370C, 15 min

StuI

Restriction enzyme                         1 ul
DNA                                                   0.4-1 ug
10X NEB buffer (“CutSmart”)        2 ul (for 1X final)
dH2O                                                  to 20 ul

Incubate 370C, 15 min

Put in separate document

Agarose Gel Electrophoresis of PCR products and RD reactions

You will perform agarose gel electrophoresis on the PCR products generated during the previous lab exercise, as well as the restriction digest reactions generated today. This will allow you to visualize your results and capture the image files for further analysis. Your reaction products will provide information that helps to confirm the presence and identity of your ‘gene of interest’ in the plasmid.

Overview

Agarose is a polysaccharide isolated from highly purified seaweed/algae. When heated to boiling in aqueous solutions, the polysaccharide molecules dissolve and fully extend; when cooled, the polysaccharide strands fold and twist with each other, forming a meshwork/gel with pores of a size determined by the agarose concentration. Dilute solutions of agarose in buffer (typically 0.7%- 2.0%) will solidify at room temp in about 20-30 min, and will form a gel, or molecular sieve with pores large enough to allow migration and separation of relatively large double stranded DNA molecules when an electric field is applied.

1 % agarose gels are good for separating DNA fragments of vs. ~250 bp- 12,000 bp (12 kb). For smaller or larger DNA fragments, the best estimation of their sizes would require agarose gels of higher percentage (with smaller pores) or lower percentage (with larger pores), respectively.

Because of its phosphate groups (with negatively charged oxygens), DNA is uniformly negatively charged at neutral to alkaline pH. As a result, once a voltage has been applied across the gel, the DNA fragments will migrate from the cathode (black) end towards the anode (red) end at a rate determined by their size- i.e., the smaller fragments will migrate faster than the larger ones. At the end of the gel run, the fragments will form a “ladder” of increasing sizes from bottom to top. DNA molecular size standards run in parallel lanes of the same gel allow an estimate of the sizes of the DNA fragments of interest.

Note:

The gels in this lab will contain ethidium bromide (EtBr); EtBr is a DNA-intercalating dye and is potentially mutagenic and carcinogenic. Wear gloves when you handle these gels, change them often (dispose of them in the designated buckets), and be aware of what you touch when you have them on. Although the running buffer (1X TBE, see below) will not have EtBr added, once the gel is placed in the buffer it (TBE) too will be ‘contaminated’ and must be disposed of in specially designated bottles in the chemical fume hood.

Reagents:

  • Samples from PCR and RD
  • Ethidium bromide (already added to the gel solution)
  • 5X stock TBE buffer (Tris-Borate-EDTA, pH 8.0)
  • Agarose- electrophoresis grade (already dissolved to 1.0%, in 1X TBE, and cast into gels)
  • Ready-to-load DNA molecular weight markers (1 kb DNA ‘ladder’)
  • 6X DNA loading dye (“6X LD”)

Lab Instrumentation:

  • Agarose gel apparatus
  • Power supply
  • Biochemi and GelDoc detection systems

For more information on gel electrophoresis, see:

http://www.dnalc.org/resources/animations/gelelectrophoresis.html

Gel Preparation: Steps 1-4 will be done prior to lab period; steps 5 and beyond are to be done by the students, after a demonstration by instructor:

  1. The gel apparatus for each team will be set up in the configuration for casting the gel: the gel tray is turned so that the rubber gaskets are in contact, making a tight seal, with the sides of the apparatus (at right angle to the orientation shown in the photo below). This configuration allows for hot liquid agarose to be poured and contained within the tray as it solidifies. (The orientation of the tray during casting is perpendicular to the axis through which the current will eventually run.)

DNA gel apparatus (orientation for run)

  1. One 10-well comb is placed in a slot at one end of the tray. Combs allow “wells” to form in the gel as it solidifies, into which samples will be pipetted. Placement of the optional comb in the central position allows for one tray to be divided into two separate “mini-gels”, which is desirable if more than 10 samples are to be run and if adequate gels” for our PCR separations.
  1. Hot (~500C) liquid agarose in 1X TBE/EtBr will be prepared prior to lab: Electrophoresis-grade agarose is weighed out and placed into 1X TBE buffer/EtBr; boiling by several short (~30 sec) pulses in the microwave is required to dissolve the agarose thoroughly, to the point where the solution is clear and no particulate agarose is visible. The agarose solution is allowed to cool to ~50- 550C before pouring into the gel apparatus. (Warping of the apparatus and damage to the rubber gaskets can result if the agarose solution is too hot.)
  1. Pouring the gel: Using a 50 ml blue-top centrifuge tube, 40 ml of the hot liquid agarose is measured and immediately poured into the prepared apparatus. The gel is allowed to solidify for ~45 min minutes, until visibly opaque.
  1. (Students) Preparation of gel apparatus for electrophoresis: After gel has solidified, carefully remove each comb by gently pulling directly upwards; try to avoid tearing the gel. Remove the gel tray from the apparatus, turn it 90° and place it so that the wells on one end will be at your left side, to be inserted into the negative (black, cathode) lead from the lid.

This is critical because the DNA, which is negatively charged, will migrate from the negative pole (black) to the positive pole (red).

  1. For each gel apparatus: Prepare an additional 300 ml of 1X TBE buffer from the 5X stock (dilute appropriately with dH2O). To mix, stretch parafilm tightly over the cylinder, and invert several times.

Buffer salts conduct electricity and keep the gel from overheating. EtBr intercolates into the DNA between the base pairs and is highly fluorescent under UV light when bound to DNA. This property aids in detection of your DNA bands on the gel (in BioChemi or GelDoc apparatus).

* Another reminder about working with EtBr (although really it is not that dangerous as long as you observe proper precautions): change gloves frequently (as needed), be mindful of what you touch, and wipe up spills promptly. “Solid” EtBr waste (including the gel itself) will all go into the designated pails; liquid EtBr waste will go into the designated bottle in the chemical fume hood.

7.  Carefully pour the 1X TBE into the gel box until the buffer reaches the “fill” line. The gel is now ready to load. (Instructor will demonstrate loading.)

Sample preparation and loading of the gel:

Note: initial plan is for 2 teams to share one gel; it is generally better to have PCR reactions on one gel, and RD’s on another gel. Plan accordingly.

DNA samples (including molecular weight standards, or “DNA ladders”) cannot be loaded directly into the gel ‘as is’; they must first be mixed with DNA loading dye (referred to here as “6X LD”) to provide the proper pH environment and density to the samples. DNA loading dyes typically consist of a slightly alkaline buffer, most often Tris-EDTA, pH ~8, to maintain the negative charge of your DNA samples (necessary for proper migration through the gel). Glycerol or Ficoll is another important component, providing density to the samples as they are loaded in the wells of the gel.

Additionally, DNA ‘loading dyes’ typically contain one or more anionic colored tracking dyes1 to assist in visualization of the progress of migration (although the dyes do NOT bind to the DNA directly).

The provided DNA molecular weight markers, or ‘1 kb DNA ladder’, consist of DNA bands ranging from 250-10,000 bp. Instructor will have the ‘DNA ladder’ already mixed with 6X loading dye to the proper concentration, and it will therefore be ‘ready to load’. You will need to load 10 µl of this DNA ladder into one lane of your gel, typically the left-most lane.

Your PCR samples will need addition of the 6X loading dye to prepare them for electrophoresis:

  1. For each of your four PCR samples, label a NEW microfuge tube. In each new tube, mix 2 µl of the green/orange “6X loading dye” with 10 µl of each PCR sample (save the remainder). This effectively dilutes the 6X sample buffer down to 1X. Mix by briefly vortexing, then pulse-spin to bring contents to the bottom of the tubes.

RD reactions (terminated with DNA loading dye) are ready to load.

Loading the gel: Place gel over blue countertop for easier visualization.

  1. For the DNA ladder: load 10 µl into the left-most lane of each gel
  2. For each PCR sample: just after the DNA ladder lane, in each subsequent lane, load all 12 µl of each prepared PCR sample. Make note of the gel loading order. It may be wise to leave a ‘blank’ lane between the two teams, if possible.
  1. For each RD sample: just after the DNA ladder lane, in each subsequent lane, load all 24 µl of each prepared RD sample from the “20 ul” reactions (with 24 ul total); for those teams with “50 ul” reactions (60 ul total), you won’t fit the entire volume into one well, so do your best to load ~30 ul per well. Make note of the gel loading order.

Again, it may be wise to leave a ‘blank’ lane between the two teams, if possible.

  1. Put on the lid, double-checking to make sure the DNA wells near the edge are at the negative (black) pole of the lid.
  1. Attach the leads to the power supply (which is turned OFF).
  1. Turn on the power supply and set the voltage to ~120 volts. The higher the voltage, the faster the gel will run (but the gel won’t turn out as well if run too fast). The voltage may be adjusted somewhat for proper timing. The gel will run somewhere between ~35-45 minutes.

Dyes present in 6X DNA LD: Xylene Cyanol FF, migrates with DNA fragments of ~4000 bp; Bromophenol Blue, migrates with DNA of ~300 bp; and Orange G, migrates with small DNA fragments of ~50 bp. You should see the dark blue dye from the sample buffer (bromophenol blue) migrate toward the positive pole.

Terminating the gel: Once the dark blue dye has migrated almost to the end of each gel, which is far enough to separate our DNAs of different sizes:

FIRST TURN THE VOLTAGE DOWN TO ITS LOWEST POINT, THEN TURN OFF THE POWER SUPPLY; disconnect the leads from the power supply and then disconnect the lid. Carefully place the gel, still in its holder, in a small plastic tray; rinse briefly with Milli-Q water (dispose of rinse water in the specially labeled bottle in the fume hood). Bring rinsed gel to the gel documentation system to visualize the DNA and obtain an image (with instructor or TA’s help).

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