Lab 3 Flashcards
Lab 1:
- Isolation and quantification of genomic DNA
Lab 2:
- gDNA quality control agarose gel electrophoresis
- gDNA quality control PCR
Lab 3:
- Restriction enzyme digest for AFLP analysis
- Adapter ligation to digested DNA
- Agarose gel electrophoresis of gDNA PCR
lab 4
- Pre-amplification
- Selective amplification
- Practice loading of an AFLP gel
lab 5
- Load and running AFLP gel
- Submit samples for capillary electrophoresis
EcoRI and MseI Restriction Enzyme Digestion of genomic DNA for AFLP
Analysis
- Do this right on entering the lab, the digest will take 2 hours and you have to do the adapter ligation towards the end of the lab.
- Your digest will contain 250 ng DNA in ≤ 18 L. You will need the genomic DNA concentration you
calculated from your PicoGreen data to determine the volume for 250 ng DNA. - Example: If your concentration was 75 μg/mL ➔ 75 ng/L
- 250 ng genomic DNA are in ……
- Pipette into a 1.5 mL micro reaction tube:
- Order of pipetting: SDW, Reaction buffer, Enzyme, DNA
- Incubate 2 hours at 37 ℃
How large are your restriction fragments on average?
What is the average spacing of restriction sites in a genomic DNA (assuming
equal content and distribution of A, G, C and T)?
Cuts every 4n bases; n = number of nucleotides in the recognition sequences
EcoRI 46 = 4096 bp ➔ cuts about every 4096 bp or with a frequency of
1/4096
MseI 4
4 = 256 bp ➔ cuts about every 256 bp or with a frequency of
1/256
What kind of restriction digested DNA
fragments can you expect?
- Both ends are EcoRI digested
5’ AATTCNNN…NNNG 3’
3’ GNNN…NNNCTTAA 5’ - Both ends are MseI digested
5’ TAANNN…NNNT 3’
3’ TNNN…NNNAAT 5’ - One end is EcoRI digested and the other is MseI digested
5’ TAANNN…NNNG 3’
3’ TNNN…NNNCTTAA 5
What is the impact of restriction sites on
fragment frequency?
EcoRI—-EcoRI fragment ➔ least abundant, average size 4096 bp
MseI—- EcoRI fragment ➔ intermediate abundant, average size between
EcoRI—-EcoRI and MseI—- MseI fragments
MseI—- MseI fragment ➔ most abundant, average size 256 bp
Control experiment: Analysis of a specific known genomic PCR fragment by agarose gel electrophoresis
gel preparation
- Prepare a 1% agarose gel. Similar to lab 2 but different agarose concentration.
- Q: Why do we use a different agarose concentration?
Q: Why do we use two different PCR samples (concentrated and ½ diluted)?
Control experiment: Analysis of a specific known genomic PCR fragment by agarose gel electrophoresis
- Run your gel at 70V constant voltage until the blue loading dye has migrated about 2/3 of the gel length.
- Stop and image the gel
- Determine the size of your PCR products using the 1kb plus ladder (Invitrogen) as a size marker
Ligation of adaptors to digested genomic DNA for AFLP amplification
- After the restriction digest ➔ heat the sample for 15 minutes at 70 ℃. This inactivates the restriction enzymes.
Q: Why? - Place on ice for 5 minutes
- Brief centrifugation to collect sample at bottom of the tube
- Add to the digested DNA
- Mix gently at room temperature, centrifuge briefly to collect contents at the bottom of the
tube - Incubate at 20 ℃ to 22 ℃ for 2 hours. The reaction will be stored for you
The T4 DNA ligase reaction
Adapter ligation solution
o 0.2 M EcoRI adaptors
o 2 M MseI adaptors
o 0.4 mM ATP
o 10 mM Tris-HCl pH 7.5
o 10 mM magnesium acetate
o 50 mM potassium acetate
diagram
slide 12
diagram
slide 13
What kind of DNA fragments can you obtain after adaptor ligation?
Circularised DNA
Circularised DNA via adapters
that were ligated to the ends
and to each other.
What double stranded products will be in the PCR tube following the a PCR
amplification if a combination of a primer that can bind the EcoRI adaptor and a primer that can bind the MseI adaptor are used?
What double stranded products will be in the PCR tube following the a PCR
amplification if a combination of a primer that can bind the EcoRI adaptor and a
primer that can bind the MseI adaptor are used?
