7.13 | Fall 2008 | Undergraduate
Experimental Microbial Genetics

Molecular Biology Boot Camp

The first two weeks of the course will be a molecular biology “boot camp” in which the whole class will conduct a short project designed to help you master some basic techniques.

SES # ACTIVITIES BOOT CAMP PROTOCOLS
1

Start 150 ml culture of E. coli + pMQ64 in LB⁺30 ug/ml gentamicin

Streak out PA14 from cryo-stock, put stock in own box in freezer

Day 1 (PDF)
2

Maxi prep of E. coli⁺pMQ64 culture

Set up digest of pMQ64 with BamHI

Phosphotase cut vector, inactivate phosphotase, freeze vector at -20°C (vector will be gel purified at same time genomic DNA inserts are gel purified)

Start overnight wt PA14, incubate in roller drum at 37°C

Day 2 (PDF)
3

Isolation of genomic DNA from PA14

Pour 2 agarose gels

Day 3 (PDF)
4

Get your DNA and it’s concentrations from the teaching staff

Set up your partial digest with BfuCI

Load and run diagnostic gel

Image 1st gel, analyze

Set up upscaled digests

Louad and run upscale gel; also run cut vector from Day 2 on this gel

Image gel and cut out correct fragment size band from gel. Freeze at -20°C

Day 4 (PDF)
5

Extraction of DNA from gel fragment

Set up ligation: do 1:3, 1:1 and 3:1. Insert vector ratios, 2 no insert controls (one for each insert sample) & a no vector control (9 ligations)

Transform ligations, do no DNA control here (cells only) (10 transformations)

**Save the rest of ligation reaction in your freezer box**

Spread x-gal on plates

Plate 2x, 150 ul (20 plates/group)

Day 5 (PDF)
6

Look at plates, calculate blue to white ratio for each ligation reaction and how many plates you need to pool to get good representation of PA14 genome in your plasmid prep.

Pool colonies from appropriate number of plates and mini prep to purify plasmid mix. Freeze prep at -20°C.

Day 6 (PDF)
7

Set up PCR on plasmid prep

Pour gel for Day 8

Play with DNAStar

Days 7-8 (PDF)
8 Run gel to check results of PCR reaction and plasmid pool library

Course Info
Departments
As Taught In
Fall 2008
Learning Resource Types
assignment Problem Sets
group_work Projects
assignment Written Assignments