bio125 yeast transformation

Preparation for bio125 transformation experiment.

2015 Feb 27 Fri, 9pm. Streaks AGY75 from Box10 G3 to SD-URA-TRP+HIS plate. Left at 30C incubator in Sci 246.

Same plate, front view

reference:
http://hongqinlab.blogspot.com/2015/01/bio125-strains-from-gammie-lab-media.html
http://hongqinlab.blogspot.com/2014/03/bio125-yeast-transformation.html

XSEDE UTK R HPC

http://www.nimbios.org/tutorials/TT_RforHPC



Video links
https://www.youtube.com/playlist?list=PLRyq_4VPZ9g_g4b3An6VTkRX_c0tAHoAj

part 1
http://youtu.be/tN9xOs91EyI

part 2
http://youtu.be/LNCtlQWehVs

part 3
http://youtu.be/C3docbZb0RY

part 4
http://youtu.be/Bv8w_H90YVQ

materials
http://www.nimbios.org/ifiles/hpc/



NimbioS,
XSEDE
UTK

bio125 Thu Feb 26, 2015 student presentations on MSH2 project

Section 1 students mostly used PPT with audio file.

Section 2 students mostly used JING.

Some students complained that teammates did not contribute enough for the presentation.

Most students did not discuss the Gammie paper, many students did not present ApE analysis on RE for WT and mutant differentiation.

Things to change in the future: Emphasize pMSH2 and pSH44 reporter plasmid.

flowchart to learn R

http://www.r-bloggers.com/how-to-learn-r-a-flow-chart/

bio125 Tue Feb24,2015 agarose gel of PCR fragment cut by RE

Section 1

Projector malfunctioned. I put my screen on GoogleHangout and posted link to News forum.

Gel were precasted for the students.

9-10am. Go over open book practice.

10am, gel picture taken.  Students are still not clear about PCR product analysis on pMSH2 during lab report.

section 1: http://spelelearn.spelman.edu/mod/quiz/view.php?id=151330

Section 2:
Projector has been rewired.

Some students were confused about the side ladder and loading dye. Luckly, we correct it before they started.

Went over open book midterm practice exam.

2:50pm. Students took pictures of their agarose gels.

CURE meeting, 20150220

Students to submit 2 pages of proposal. For 6-week.
Full time, 8-5pm, on Spelman campus.

“Research project for students” (housing on campus $800).
Experimental expense $500.

Proposals should be written by students, but in ‘consultation’ with faculty mentors.

Not necessarily A students, but motivated students. Goal for PHD or engineering degrees in the end.

No stipends for faculty mentors. (Support used for postdoc).

1.7M for HBCU CURE grant.

Basically Summer REU (not course-based).

Pam Stegall as program coordinator for HBCU CURE.

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.


Skipped: 
In class mutant analysis

MCAT exercises

Cognant sites between human and yeast MSH2

Design mutagenic primer

Design mutagenic primers
https://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

Ref:
http://hongqinlab.blogspot.com/2014/02/bio125-re-of-pcr-fragment-to-verify.html

fye, mashmallow test

mashmallow test youtube video

socrative short answers

weka, lifespan prediction

https://weka.waikato.ac.nz/dataminingwithweka/preview

toread, dN/dS

dN/dS, scaled selection coefficient

http://mbe.oxfordjournals.org/content/early/2015/02/13/molbev.msv003

Proof and simulation based.

PAML is highly biased with the synonymous codon has different fitness.

bio125, Feb 17 Tue 2015, PCR exercise (lab postponed due to icy weather)

Section 1: class canceled. 2014 midterm exam in class time.

Section 2:
1:10pm, class started.

by 1:50pm, go over PCR case study problem on pin3. The last question has not enough information. One of the choice is also wrong, because heterozygous should give 2 bands. 

1:50pm - 2:50 pm. 

In class mutation identification exercise. Some students did not know which tools to use. 

ApE primer analysis

2:50-3:20pm. MSH2 project overview. 

3:20-3:30 MCAT PCR exercise. 

Asked a student to summarize the class activities.

Presentation ended in group 1.

Not used. 

Cognant sites between human and yeast MSH2
Design mutagenic primer

Design mutagenic primers
https://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

Review bio125 midterm exam from previous years

Postponed RE cut of PCR fragments
Ref:
http://hongqinlab.blogspot.com/2014/02/bio125-re-of-pcr-fragment-to-verify.html

A tour of machine learning algorithms

http://www.datasciencecentral.com/profiles/blogs/a-tour-of-machine-learning-algorithms?xg_source=activity

internal speaker disppeared in Yosemite

Bugs: Internal speaker disappeared in Yosemite, after using external speaker
Ace laptop, Yosemite
Internal speaker disappeard on "Sound preference"->"Output" menu. No sound played.

Solution: Plug and unplug speaker input, and really jiggled it a bit to flip the switch. 

http://betanews.com/2014/08/26/how-to-fix-disabled-audio-in-os-x-10-10-yosemite-beta/

https://discussions.apple.com/thread/5432158

2015 Research Day announcement

As this year's co-chairs, it is our distinct pleasure to announce Research Day 2015.  The event will occur on Friday, April 17th from 8:30 AM to 5:00 PM.  The theme for this year is "Inquire, Discover, Imagine, Explore, Create".   Research Day is one of the liveliest and most intellectually engaging events in the Spelman College academic year. It showcases our students' scholarly and creative work across all academic disciplines. Please encourage your students to submit abstracts/summaries of their work by the deadline: 11:59 PM, Sunday, March 3rd, 2015.Abstracts are limited to 250 words. The following link may be used for submissions effective today:

                                             http://princess.spelman.edu/researchday.nsf

Please note the following key points for faculty and staff members: 

-        If you have advised a student on a project, please encourage her to submit an abstract/summary of her work for Research Day.  Please work closely with your student in the development and proofreading of her abstract. 

-         If you are the advisor to a student’s project, you will receive an email requesting your approval of her abstract once she submits her work.  You will need to do this through Lotus Notes.

-        Please ensure that you have space in your email to receive notification that your student has submitted an abstract. The deadline for faculty advisors' approval of student abstracts isMarch 3, 2015. 

- Please announce Research Day 2015 to all of your students.

Research Day 2015 is an opportunity for students to showcase their academic achievements. For many, it is one of the first steps they take while at Spelman College to demonstrate inquiry-based scholarly excellence.  We encourage each student to participate in Research Day 2015, and consider this as a true Gateway to Excellence within her chosen field.

Min-protein pole-to-pole oscillation and E coli cell division

Soft matter, 2014, 10, 2388, Hoffmann, Schwarz,

Oscillations of Min-proteins in micropatterned environments: a three-dimensional particle-based stochastic simulation approach

"We performed our simulations with Smoldyn 2.28 which is a particle-based stochastic simulation

tool for reaction-diffusion systems in user-defined geometries that is freely available at http://www.smoldyn.org.  This simulation framework has been successfully applied to many

different biological systems, for example to protein diffusion between two electrofused cells.31 Especially due to its versatility regarding geometries and reactions on 2D surfaces embedded in

a 3D environment"

bio125, Thu, PCR reaction, past exams, restriction enzyme lab report

Section1: 
8-8:45am Go over PCR protocols, using R code for dilution. Make master screen cast.
Group 2 worked out the master mix on the board.

Each rxn: 2 uL of primer mixture and 23uL water for each reaction + 1 bead

                 2 uL 5n/uL template

8:45-9:30am. students diluted their plasmid stock solution to 5ng/uL as PCR templates.

by 9:30 Setup PCR. (Warning: Glove and PCR bead can attract with electrostatic pull)
First group finished setting up PCR, but some groups still work on the template dilutions.

I asked the leading group to follow youtube video and work on primer analysis.

10-10:30am 

Screen cast:
PCR primers on pMSH2, followed by restriction enzymes
https://www.youtube.com/watch?v=aE8UUGJYzHw&feature=youtu.be
Problem: some students choose 'circuar' for PCR framgment.
ApE PCR primer analysis and followed by restriction enzyme, gel simulation

Announcement:
 Go over lab report:  RE lab report will be due, video and R code

Section2: 
Only five students were in by 1:10pm.

1:15pm start class.

2:10pm students started setting up reactions.

by 3pm. video to use provided RE to analyze PCR fragments of WT and msh2 mutants.

Unused and Moved to next week

MCAT PCR questions

PCR_inclass_data analysis
BLAST

Past exam questions on gel pictures.

todl Design site-directed mutagenesis primers

todo, rescue plasmid from yeast, bio125

http://www.clontech.com/US/Products/Protein_Interactions_and_Profiling/Yeast_Plasmid_Isolation_Kit/Plasmid_Minipreps

todo, bio125, mutagenic primer design

Make a video using the following link

http://hongqinlab.blogspot.com/2014/04/design-mutagenic-primer-to-engineer.html

Respondus, converting old exams to Moodle exams

Instead of modify the word format, I found it is more convenient to load the WORD file into Respondus directly, and "preview"-> "modify".

todo: biomedical engineering in biology major

  Biomedical biology engineering 

bio125 Tue, Feb 10, 2015, PCR principle

Before class: 
1) color-print PCR paper exercises
2) MCAT PCR questions

Section1:
8-9:30pm, in class ApE and identify mutation. Identify mutation in each group.

30 minutes PCR paper exercise: ask student to write PCR procedure first.

20 minutes, Analyze gel pictures. Example of how to label a gel picture.

last minutes, asked students to go around and recap what have learned.

Section2:

1-2pm, went over geneX exercise

2-2:44, identify mutant in each group. 

2:45-3:20 PCR paper exericse. 

Lab instructor observed that some students try to put both forward and backward primers on the same template. 

Did not have time for gel analysis for section 2.

Students came out of a chemistry exam and claim an open book component will be due by 6pm. In the bio125 class, many students were busy working on their chemistry exam. 

Review RE exercises

Show a youtube video. I can ask one student group work on the board with tapes and markers.
https://www.youtube.com/watch?v=_YgXcJ4n-kQ
Materials: Razors, scotch tapes, color markers

PCR MCAT questions

Past exam questions on gel pictures.

Thu:
Design site-directed mutagenesis primers

Reference:
http://hongqinlab.blogspot.com/2014/02/bio125-re-of-pcr-fragment-to-verify.html

online problem sets

Careful checking of online activity log show that some students did not download the required R codes, but was able to get 100 points on the quiz. This indicates that they took advantage of the multiple attempts, and systematically tested the answers.

This did not align with the intent of assisting student learning.

bio125 Thu, Feb 5, 2015, gel electrophoresis

Section 1:
37C incubation

by 8:15, reviewed agarose gel

8:30 I did a demo. R code on RE recognition site. Length of DNA words.

http://youtu.be/U1EdV5liCok

9am paper exercise on RE site, compatible RE enzymes. (This is a question in restriction enzyme assignement)

https://www.youtube.com/watch?v=W_rgsBz12Tk

x Paper exercise using grid paper on RE compatible sites

9:10 started pouring gel. Experiment of gel electrophoresis

Every bench has a gel box and power set. Students were asked to pour their own gels.
35mL of Gel-EB mix in falcon tube in 60C water bath. Kioko said 35mL gave 0.5cm depth of well that can hold upto 15uL sample.

Make sure DNA run to the positive side, put wells on the black (negative side).
Make sure power setting is on Volts. Set to 120Volts. (VWR power setting is default to Volt).

The dye is 6X, so 2 uL + 10 uL sample.

by 9:38am. Many students have pour gels. I asked them to work on Rstudio exercise on DNA words.

by 9:50am. Gel are set up, buffer are poured.  DNA ladders in 5uL were given to students.

by 10:05pm, most group finished gel loading.

Ask one member of each group to stay after class to take picture of the gels.

10:20 mcat. I finished two questions and ask students to work on them after class.

10:45am. Lab instructor used  a hand-hold UV lamp.

Section2;
1:05pm, gave mcat restriction enzyme questions to student. Socrative quiz.
  Q: blunt ends and sticky ends

Paper exercise

1:44 R DNA word video from morning

by 2:33, finish paper exercise and sticky ends.

2:40  Gel is ready, buffer are poured. Student spin down the samples and started to gel running.

2:55pm gel loading started.
(Tip: Size ladder was given to students after loading dye have been used, thus avoiding disastrous mixing up that happened in the past).

Students ask why we have to run agarose gels:
1) Check plasmid, are they what we want?
2) Check plasmid concentration.

Students have 4pm bioseminar. So, one of them has to come back to take pictures of the gels.

At 5pm, students came back to take pictures.

Not used:
=============

MCAT questions on RE

Ruler for gel measurement. standard curve exercise again.

ApE exercise. Most students

GoogleDoc final project report

=============

Identification of mutation

Protein alignments

PCR principle

Cognant site, identification of mutations

week 2/2-2/6, 2015, plan for week 4:

day 1:

pre-class

overview for MSH2 (single, DW), chapter 20(genetic technology): restriction digestion (DW), molecular cloning (DW), pre-lab, build the math into it (DW)

In class:

digestion, XbaI ApaI, labnotes (DW), presentation on MSH2.

ApE_restriction enzyme&gel simulation, Video (HQ),

Rstudio on receipe (Qin)

Rstudio on RE site recognition, longer RE site should have few occurrence in sequences. permutation of all possible sequences given the number of basepairs  (Qin)

day 2:

pre-class:

electrophoresis (DW), pre-lab (DW),

in-class:

MCAT (HQ)

running gel (maybe do standard curve) (DW)

R code and standard curve (Qin)

data analysis (DW) as a backup activity

R code, generate sequences for PCR paper exercises

For bio125 , paper PCR exercises


# Generate sequences for PCR paper exericses

library("seqinr");

list.files()

# Load plasmid pMSH2 sequence into R
seqs = read.fasta( "pMSH2.seq")

# look at the first sequence
seq1 = seqs[[1]]
seq1 = toupper(seq1[1:91])
seq1RC = toupper(rev(comp(seq1)))

c2s(seq1) #sense strand
c2s(seq1RC) #antisense stand

FYE, procrastination

http://youarenotsosmart.com/2010/10/27/procrastination/

ulyssis bergain

FYE netflix watch list

there were only fifty students in the crowd, many watched their cell phones or chatting among themselves. in genreal, students in the back did not pay attention, those at front participated.

thirty minutes

mischels marshmallows radiolab.

An faculty walked to the back and quieted the chatting.

R plot of population heterozygoy

https://github.com/cooplab/popgen-notes/blob/master/Rcode/Loss_of_heterozyg_varying_pop.R

roll verification

Date: January 28, 2015

RE: Verification of OFFICIAL class rosters for spring 2015

Federal regulations mandate that we verify that all students attending classes are actually enrolled.  Additionally since we have not reached our enrollment target of 1960 (currurently 1940), we need to identify students who are attending classes but have not fulfilled their financial obligations to the College.   We may be able to help some of these students, so please help us to identify them.

To verify class rosters, please use the following instructions for each of your classes:

· Log into Banner web· Go to Faculty and Advisors
· Select Midterm Grades
· Select the term -  spring 2015
· Select course to verify enrollment
· In the column ATTEND HOURS

• Place a zero (0) if student has never attended; these students will be dropped from the class. No record of their enrollment is recorded.
• Place a one (1) if the student is on the roster but has stopped attending; these students will be administratively withdrawn, having a “W” on their transcripts.
•  If a student is attending class but not on the roster, please reply to this email with the student’s name, 900#, and course title andsection, if there is more than one section of the same course.

Please complete your course verifications on or before February 5th.  The sooner the better since we are trying to help register students.

bio125, Tue, Feb 3, 2015

Section 1:
8-9:30am go over protocol. Mater mix was not understood.
I went with R code on digestion recipe

9:30-10:30am. Student started dilution DNA. 

Lab instructor took the original plasmid stock away as soon as student finished the dilution. 

Problems: 

_ most students have trouble with maxter mix recipe

_ some students shot 8ul in the air to the tube

_ students are confused by the serial number and plasmid names

Section 2:
1-2pm. went over protocol.

2pm. Student started to make DNA dilutions.

by 2:20pm. I went through master mix recipe.
by 3:10pm. Most student are done with reaction setup.

3:12pm, lab note 3 Moodle online assignment

=== thing postponed 

ApE exercise
Dilute plasmid DNA, label tubes group number, plasmid, concentration

GoogleDoc final project report
R code on RE recognition site
MCAT questions on RE

RE paper exercise,

=============
Ruler for gel measurement

Identification of mutation

Protein alignments
Gel electrophoresis

PCR princple

Cognant site, identification of mutaitons

week 2/2-2/6, 2015, plan for week 4:

day 1:

pre-class

overview for MSH2 (single, DW), chapter 20(genetic technology): restriction digestion (DW), molecular cloning (DW), pre-lab, build the math into it (DW)

In class:

digestion, XbaI ApaI, labnotes (DW), presentation on MSH2.

ApE_restriction enzyme&gel simulation, Video (HQ),

Rstudio on receipe (Qin)

Rstudio on RE site recognition, longer RE site should have few occurrence in sequences. permutation of all possible sequences given the number of basepairs  (Qin)

day 2:

pre-class:

electrophoresis (DW), pre-lab (DW),

in-class:

MCAT (HQ)

running gel (maybe do standard curve) (DW)

R code and standard curve (Qin)

data analysis (DW) as a backup activity

PCA, snp matrix, human population

For human snp in maxtrix

                  site1   site2
people1      0          1...
people2      2          0 ...

0: aa, 1 Aa, 2 AA

So, PCA can be calculated from this matrix pretty straightfoward.

Ref
https://www.youtube.com/watch?v=SlnOxHhypDg

admixture mapping

admixture mapping
two ancestral population

https://www.youtube.com/watch?v=syKWlFjzUss

https://www.youtube.com/watch?v=zGha8i8Xzpk

Chronicles Faculty salary data

http://chronicle.com/article/2013-14-AAUP-Faculty-Salary/145679#id=table

bio125 R: sequence exercise, DNA words, RE sites

Files nucleotid_word_RE_demo.R

# Exercise to study how occurence of DNA words are influenced by their length.
# What are the occurence of 1-letter, 2-letter, 3-letter, ... 8-letter DNA words? 
# Learning outcome: longer words should have less occurrence in DNA
# by Hong Qin, Jan 30, 2015, for Bio125 @ Spelman College

library("seqinr");

#reverse complementary DNA in R
c2s( rev(comp(s2c("atg")) ))
c2s( rev(comp(s2c("atcg")) ))
c2s( rev(comp(s2c("gaattc")) ))

# Try on your own
# read in some bacterial 16s rDNA sequences
# seqs = read.fasta( "http://www.bioinformatics.org/ctls/download/data/16srDNA.fasta",seqtype="DNA");

# Use plasmid pMSH2 sequence
seqs = read.fasta( "pMSH2.seq")

# look at the first sequence
seq1 = seqs[[1]]
seq1    #sequence in array
seq1RC = rev(comp(seq1))  #reverse complement
  
c2s(seq1)  #print out sequence in a long string
count(seq1, 1) #nucleotide composition
count(seq1RC, 1) #nucleotide composition
mean( count(seq1, 1) )

count(seq1, 2) # occurence of two-letter DNA words
count(seq1RC, 2)
mean( count(seq1, 2) )


count(seq1, 3) # occurence of 3-letter DNA words
mean( count(seq1, 3) )
results = count(seq1, 3)
results[c('att','aat')]

results = count(seq1RC, 3)
results[c('att','aat')]

# ?? # occurence 4-letter words?

results = count(seq1, 4)
results['agct']

results = count(seq1RC, 4)
results['agct']



# ?  # occurence of 5-letter DNA words

# ? # occurence of 6-letter DNA words
# results = count(seq1, ?? )

# EcoRI site is GAATTC
# How many GAATCC in pMSH2? 

results = count(seq1, 6)
results['gaattc']

results = count(seq1RC, 6)
results['gaattc']


count(seq1, 8) # occurence of 8-letter DNA words
mean( count(seq1, 8) )
median( count(seq1, 8) )
max( count(seq1, 8) )
hist(count(seq1, 8), br=30)

results = count(seq1, 8)
#NotI site is GCGCCGC
results['gcggccgc']

results[results==1]