Thursday, February 19, 2015

bio125 Feb 19, Thu, digestion of PCR fragment of msh2 mutant genes

Before class:
Set 4 water bath/blocks: 60C, 55C, 37C, 25C

NEB reformulated their buffers. 
Buffer 2.1 = old buffer 2 + BSA

During the lab, the Lab instructor will give buffer and BSA to students. The students will go to the lab instructor, asking for the specific enzyme that they need. 

Section 1:
pre-class assignments
Average is more than 90% for PCR-case studies, ApE PCR primer analysis, BLAST assignment.

8-9am, go over prelab assignment, protocol.

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

todo: announce presentation next week, and moodle submission link.

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.

In class mutant analysis

MCAT exercises

Cognant sites between human and yeast MSH2
Design mutagenic primer

Design mutagenic primers
Learning objectives: Cognant site between human and yeast, preferred codons, primer design


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