Lab 9 Finish Genotyping - Running PCR products on a gel

Gel electrophoresis (DNA separation)

A technique where DNA fragments are pulled through a gel matrix by an electric current; for DNA, an agarose gel is used. Larger fragments travel more slowly through the small pores in the gel matrix, so DNA separates primarily by size

Why DNA separates by size (not charge) in agarose gel electrophoresis

The buffer (TBE or TAE) is slightly basic and neutralizes the charge of DNA, so dsDNA fragments are primarily separated by size and not charge

Agarose

A polymer of galactose extracted from seaweed; used to make gels for DNA electrophoresis. The concentration affects resolution: higher agarose content allows smaller band differences to be resolved (up to 3% for small fragments)

DNA ladder

A sample containing bands of known DNA sizes that is run alongside experimental samples on a gel. The ladder allows determination of the size of fragments in PCR samples by comparing migration distance. We use NEB's 1kb plus DNA ladder

Ethidium bromide

A commonly used dye to visualize DNA on gels; intercalates into DNA and fluoresces when illuminated with UV light. It is a carcinogen because it intercalates into DNA

SybrSafe

A DNA intercalator (inserts between base pairs) used to visualize DNA on gels. When illuminated with blue light, it emits green light. Although billed as safe, it intercalates into DNA so gloves should be worn and handle with caution

Loading dye (gel electrophoresis)

Contains sucrose (to make samples sink to the bottom of wells) and visible dyes (bromophenol blue and xylene cyanol, which looks pink). The dyes run at predictable locations so you know when to stop the gel

Why add loading dye to PCR samples

The sucrose makes samples sink to the bottom of the well, minimizing samples floating between lanes. The visible dyes allow monitoring of gel running progress

How to determine when to stop a gel

Run the gel long enough so bands of interest separate, but not so long that desired products run off the bottom. The pink dye (xylene cyanol) can be allowed to get close to the bottom of the gel

Gel electrophoresis buffer types

TBE or TAE; the running buffer must be the same buffer used to dissolve the agarose. Add enough to just cover the gel surface and fill wells (too much buffer channels current over the gel, increasing run time)

Gel electrophoresis voltage and time

Run at 120 V for approximately 25 minutes minimum. Each gel is idiosyncratic; monitor the dye front to determine when to stop

Why photograph gels before leaving lab

At room temperature, small PCR products will diffuse through the gel over time, causing bands to lose sharpness. View and photograph immediately after running

How to interpret gel results for CRISPR editing

Lanes with 2 bands (wild-type size and insert size) = heterozygous for insertion. Lanes with one band larger than N2 band = homozygous for insertion. Wild-type N2 control shows only the smaller wild-type band

What to do if a worm is homozygous for the insertion

Chunk a small number of progeny from that plate onto a fresh plate. Put a red star on the plate, label with section, initials, date, and "homozyg unc-32::gfp." Keep both plates at 15°C for further study (will need sequencing to confirm)

Why we single non-Unc worms for genotyping

The repair template contains wild-type unc-119; injected worms are unc-119(ox819) (Unc phenotype). Worms that acquired the repair template will be non-Unc, making them candidates for successful CRISPR editing at unc-32

Gel electrophoresis of heterozygous vs homozygous insertion

Heterozygous: two bands (wild-type band and larger insertion band). Homozygous: one band, larger than wild-type (both chromosomes have the insertion). Wild-type: one smaller band

What to do if a gel lane has no bands or smeared bands

Possible causes: too much sample loaded (sample near top of well runs differently than sample near bottom), PCR failed, or no DNA template. Smearing can also occur if gel was poured too thickly or run too long

Relationship between log(bp) and migration distance

If you plot the distance a DNA strand migrates relative to the log of the size of a band, you get a straight line. This allows size determination of unknown bands using a ladder standard

Why agarose is used for DNA but polyacrylamide for proteins

DNA is larger than proteins (3 nucleotides = ~1,000 Da; 1 amino acid = ~110 Da). Agarose pore size is better suited for separating large DNA molecules, while polyacrylamide is better for smaller proteins

What to record about gel in notebook

Percent of agarose poured (e.g., 1%) and whether it was buffered by TBE or TAE. Also record which lanes correspond to which samples

How to pour an agarose gel

Seal gel tray (with black barriers or autoclave tape), insert comb at one end (DNA is negatively charged, so wells go at negative/black electrode end), pour melted 1% agarose-TAE-SybrSafe solution to cover about 1/2 the height of comb teeth (do not pour too thickly), avoid air bubbles, allow 20 minutes to solidify

Why check agarose before pouring

Look for lumps; if agarose is lumpy (from sitting out too long), remelt it. Do not pour a gel with lumpy agarose because it will run poorly

How to load samples on a gel

Add loading dye (6x) to samples: either 2 μL dye + 10 μL sample (or vice versa) in fresh tubes or on parafilm. Load into wells, being careful not to overfill (sample near top of well will run differently than sample near bottom)

How much ladder to load

NEB 1kb plus ladder concentration is 0.1 μg/μL; recommended load is 0.5 μg, so load 5 μL (0.1 μg/μL × 5 μL = 0.5 μg). Loading dye is already added to the ladder

where is DNA loded

Why DNA is loaded at the negative (black) electrode

DNA is negatively charged (due to phosphate backbone), so it migrates toward the positive (red) electrode. Wells are placed at the negative end so DNA runs through the gel toward the positive end

Why the 500 bp band in NEB 1kb ladder is thicker

The 500 base pair band is somewhat thicker than surrounding bands to serve as a landmark. The 1 kb and 3 kb bands are the brightest

What to check after removing comb from gel

If ripping the gel, gently rock the comb back and forth slightly before pulling straight up in a continuous motion to avoid tearing wells

What the two dyes in loading dye indicate

Bromophenol blue and xylene cyanol (pink) run at different rates depending on agarose concentration and buffer. Use their positions to estimate how far DNA of different sizes has migrated

Predicted PCR product size in unaltered worms vs worms with GFP insertion

Determine by identifying where primers bind in unc-32 sequence (using Word search, NCBI Primer BLAST, or BLASTn). Add the size of the GFP insertion (from repair template) to the wild-type amplicon size for edited worms

What the gray box and blue box in repair template represent (gel context)

The homology arms (60 nucleotides each) that match the target DNA sequence adjacent to the DSB; these flank the GFP and unc-119 insertion

Primer binding site determination methods

Use Word search tool and word count tool; use Google to create complement or reverse complement of sequences; use NCBI Primer BLAST; or use NCBI BLASTn restricted to highly similar sequences (mega BLAST)

What to do if a student did not have bands on their gel

The class will be posted a sample gel to analyze. Indicate band sizes in ladder and sample bands, and label size of sample and ladder bands

Molecular weight comparison: 3 nucleotides vs 1 amino acid

3 nucleotides of DNA has higher molecular weight (~1,000 Da) than 1 amino acid (~110 Da). DNA nucleotides are larger than amino acids

When determining amplicon size, do you include primer binding sequences

Yes, the amplicon includes the sequence that primers bind to because the primers become part of the amplified product (they are extended by polymerase to include the complementary strand)

What the ladder in question 4 represents (not the NEB ladder)

A different ladder than the one used in the experiment; used for hypothetical illustration of heterozygous vs homozygous band patterns

Expected gel pattern for 400bp wild-type with 150bp insertion

Heterozygous: two bands at 400bp and 550bp. Homozygous: one band at 550bp. Wild-type control: one band at 400bp

What the crRNA sequence indicates for PAM location

crRNA: GAGAATCTCTAA GATCACCT. The PAM is the sequence adjacent to the target site; in this experiment, the PAM was likely near the insertion site at the 3' end of unc-32

Why we keep plates of worms homozygous for an insertion

These are good candidates for further study. They will be sequenced to ensure the repair occurred as intended, and can be used for downstream experiments to visualize UNC-32::GFP localization

What the 1% agarose concentration means for resolution

1% is relatively low concentration, meaning larger DNA fragments can be resolved but small size differences may not be distinguishable. Higher percentages (up to 3%) are used to resolve small size differences in small fragments

Why students should pour their own gel if they haven't before

To gain experience with casting gels, which is a fundamental molecular biology technique. Students who have poured gels before can team up with those who haven't

What to do with leftover PCR samples after running gel

Keep them in case the gel needs to be re-run or if confirmation is needed; labeled tubes can be stored at 4°C or -20°C for future analysis

Why the gel doc tray holds 2 gels

To allow two groups to view their gels simultaneously, saving imaging time. Students can use phones to take photos of the gel image off the geldoc screen (student not wearing gloves should take the photo)

What "chunking" means

Transferring a small piece of agar with worms from an original plate to a fresh plate; used to propagate a strain without picking individual worms. Used when a worm is found to be homozygous for the desired insertion

Why sequencing is needed after identifying a homozygous insertion worm

To confirm that the repair occurred as intended (i.e., the GFP tag was inserted correctly in-frame at the C-terminus without unintended mutations). Gel electrophoresis only shows size change, not sequence accuracy