Run restriction digestion: TA's will take the pictures for you.
Review some points
Sequencing techniques
Analyze sequencing data
Report
Calculating the volume of miniprep plasmid DNA solution for your restriction assay
Absorbance (4μl in 400μl water) = 0.05
Concentration = 0.05 * 50 (μg/ml) * 100= 250 μg/ml =250 ng/μl
c1*v1 = c2 * v2
1's = stock solution
c1= 250 ng/μl
v1= ?
c2 = 6 ng/μl (you needed 300ng in 50 μl= 6 ng/μl)
v2 = 50 μl
v1= 6*50/250 μl = 1.2 μl
Schematic of the map-based cloning process.
Jander G et al. Plant Physiol. 2002;129:440-450
©2002 by American Society of Plant Biologists
TAIR worksheet: 8. Which are the genes on either side of GA20OX-1?
TAIR worksheet: 8. Which are the genes on
either side of GA20OX-1?
AT4G2510
TTA
AT4G25420
CAT
ATG
AT4G25430
TGA
Automated dideoxy DNA sequencing with fluoresncent ddNTPs
http://www.mun.ca/biology/scarr/Fluoresc
ent_didoxy_sequencing.html
http://www.dnassequencing.com/2011/01/
07/automated-dna-sequencing-6/
Next generation sequencing: Illumina/Solexa sequencing
Next generation sequencing: Illumina/Solexa sequencing
Advantages:
Fast
Cheap (overall)
Can be used for RNA sequencing and quantification
Disadvantages:
Sequence reads are short
BLAST
The Basic Local Alignment Search Tool (BLAST) finds
regions of local similarity between sequences. The program
compares nucleotide or protein sequences to sequence
databases and calculates the statistical significance of
matches.
BLAST can be used to infer functional and evolutionary
relationships between sequences as well as help identify
members of gene families.
http://blast.ncbi.nlm.nih.gov/blast.cgi