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
