Set 4 water bath/blocks: 60C, 55C, 37C, 25C
NEB reformulated their buffers.
Buffer 2.1 = old buffer 2 + BSA
Section 1:
pre-class assignments
Average is more than 90% for PCR-case studies, ApE PCR primer analysis, BLAST assignment.
9-10:15 am, students start to work on master mix,
One student asked why we use XbaI-ApaI to cut plasmid DNA, but use different enzyme to cut PCR fragments.
Problem: it looks students are confused by 20X BSA ad hoc method. I should add BSA directly into the master mix.
I reviewed 2014 midterm exam during the lab period, while the lab instructor worked with the students.
Problematics topics in 2014midterm practice:
-> #24, Which statement about mismatch repair is 'incorrect'? (This kind of negative argument always seem to be a problem).
-> #32, agarose gel and plasmid RE map
-> #28, pMSH2 plasmid
-> #35 Standard curve + dilution (straightforward standard curve is actually 100%, but the extra dilution tripped half of the class).
-> #30, RE compatible sites
->#27, pMSH2 plasmid
-> #10 cDNA and RE sites
->#14, types of mutations
->#31, primer concentration analysis
Section 2:
1pm -1:30 , video section 1, go over 2014 midterm exam,
1:30pm, go over master protocol.
Tip: Master mix like the main ingredient for cup cake. PCR products are like different toppings for each paper cup.
Review 2014 midterm exam
Most of the section 1 problematics ones also occur in section 2.
by 2:50pm. Several groups finished experiments. I ask them to finish online notes for lab 6, and the work on 2014 midterm exam.
Skipped:
In class mutant analysis
MCAT exercises
Cognant sites between human and yeast MSH2
Design mutagenic primerhttps://www.youtube.com/watch?v=Qy_Z2H_KAwo
http://hongqinlab.blogspot.com/2014/04/design-mutagenic-primer-to-engineer.html
Learning objectives: Cognant site between human and yeast, preferred codons, primer design
http://hongqinlab.blogspot.com/2014/02/bio125-re-of-pcr-fragment-to-verify.html
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