Biology 475 - Molecular Biology Lab
Plasmid Isolation & Sequence Reaction Set-Up
Goals
Understand the origin and identity of your plasmid and the 16S insert
Understand how to isolate high-quality/sequencing-grade plasmid with 16S insert
Master di-deoxynucleotide-based sequence analysis
Retrieving 16S Genes From Communities
Extract TOTAL DNA from Yellowstone layers - DONE, you will repeat in second half of term
PCR-amplify 16S genes from DNA = INSERT - DONE, you will repeat in second half of term
Clone 16S genes into E. coli using plasmids - DONE, you need to understand for this lab
Screen clone libraries 16S gene populations - DONE, you just did comparable fingerprinting
Sequence 16S rRNA and use software to identify - You will be doing
Our E. coli Cloning Vector - PCR/TOPO-T/A (4 kb)
Origins of replication - E. coli and yeast
Selectable antibiotic resistance gene - ampicillin
Purchased “open” with T/A overhangs and TOPO - pastes in products (or sometimes self)
Cloning sites flanked by EcoRI sites - Understand provided data about class clone library
During the pre-lab discussion, we will review how these clones were screened so you can see
the data that gave rise to your clones.
PLASMID/CLONED DNA ISOLATION ACTIVITIES
Each Person Will Receive 2 Culture Tubes
Each tube has 4 ml E. coli grown 12-16 hours overnight in LB media with ampicillin
Each E. coli clone harbors a different 16S insert in its plasmid
Your job will be to isolate high-quality, sequencing-grade plasmid DNA
You will be judged on the quality of your DNA in two ways: (1) your DNAs will be run on agarose and I
will judge it for quantity/quality; (2) if it is a poor preparation, your sequencing reactions will not work
Production of Cleared Lysate - You Should Be Done With This By 1:30
Obtain 2-3 2 ml centrifuge tubes and label with your clone numbers on the lids
Carefully pour 2 ml each culture into a 2 ml centrifuge tube and spin 10K for 5 minutes
Pour supernatant into autoclave waste bucket
Pour remaining 2 ml culture into the correct centrifuge tube - repeat above spin
Pour supernatant and blot inverted tubes on a paper towel 1-2 minutes to remove excess media
Add 250 ul Cell Resuspension Solution (Tris-HCl, EDTA, RNAse) and pipette to resuspend thoroughly
Add 250 ul Cell Lysis Solution (NaOH, SDS) and firmly invert 4X; set aside 1-5 minutes until clear
Add 10 ul Alkaline Protease Solution and firmly invert 4X; set aside 5 minutes
Add 350 ul Neutralization Solution (potassium acetate, acetic acid) firmly invert 4X (observe precipitate)
Centrifuge 14K 10 minutes to pellet precipitated proteins, membrane, carbohydrates, genomic DNA…
While spinning, insert Spin Column into 2 ml Collection Tube - the latter contains DNA-binding
substrate that will purify crude lysate. Prepare as many apparati as you have clones, LABELING the
spin column with the clone number!!!
DNA Purification From Cleared Lysate - You Should Be Done With This By 2:15
CAREFULLY transfer 800-850 ul cleared lysate/supernatant into spin column - AVOID precipitate
Centrifuge 14K for 1 minute; discard flow-thru (it has all non-plasmid contaminant)
Replace empty Collection Tube and add 750 ul Column Wash Solution (mostly ethanol, some EDTA)
Centrifuge 14K for 1 minute; discard flow-thru
Replace empty Collection Tube and add 250 ul Column Wash Solution
Centrifuge 14K for 2 minutes; discard Collection Tube - replacing with sterile 1.5 ml tube (cut off lid)
Elute plasmid by adding 100 ul nuclease-free water to Spin Column; centrifuge 14K for 1 minute
Transfer 100 ul pure plasmid to well-labeled 0.65 ml centrifuge tube - place on ice and proceed
While you are setting up your sequencing reactions, 5 ul of each of your preparations (plus 2 ul loading
dye each) will be loaded on a 1% agarose gel and judged for quality – uncut.
DNA SEQUENCING - PART ONE
ASSUMED BACKGROUND
Review Replication in vivo
DNA Sequencing - contrast…
Template = 1 genome
Template = millions of copies of gene
Start = origin/RNA primer
Start = man-made fluorescent DNA primer
Unwound by helicase
Unwound by boiling
Enzyme = DNA Pol
Enzyme = Taq, actually… PCR-based
Monomers = all dNTPs
Monomers = mix of dNTPs and ddNTPs
Products = two full-length genomes
Products = millions of random length products
Fragments representing various lengths of the ENTIRE target sequence can be generated this way.
This is why you need millions of high quality starting templates. In reality, four reactions are set up one with only ddATP, one with only ddTTP, etc.
Sequencing Kit Background
As recommended by our Li-Cor DNA Sequencer, we utilize the Epicenter Sequencing Kit (100
reactions = $250). This kit contains the following parts that you should understand:
Cocktail: Taq Pol, Buffer, and Primer*
Stop G Mix: ddGTP plus dNTPs
Stop T Mix: ddTTP plus dNTPs
Stop A Mix: ddATP plus dNTPs
Stop C Mix: ddCTP plus dNTPs
*We buy a fluorescent primer that binds to a
specific portion of the vector next to the cloned
insert; you will receive cocktail that has been
mixed with primer.
Sequencing Reaction Set-Up
For each clone, set up 4 STRIP TUBES and one SAMPLE tube. Label the strip tubes by writing the
clone number on the neck of the FIRST TUBE ONLY. Orientation is EXTREMELY important!
Add 2.0 ul of the Stop T Mix to the BOTTOM of the FIRST tube; 2.0 ul Stop G Mix to the next, A Stop
Mix to the next, and C Stop Mix to the last. Again, the order (T, G, A, C) is EXTREMELY important!
You may want to write which stop solution went in which tube (on the neck) just in case. Place on ice.
In the ice bin at your table, you will find a tube labeled "cocktail." Add 9.7 ul cocktail to your SAMPLE
tube. Add 7.3 ul of your plasmid to your SAMPLE tube - gently pipette up and down to mix.
CAREFULLY add 4.0 of the mixed cocktail/sample to each of the four STRIP TUBES. Use a different
tip each time and MAKE SURE YOU PHYSICALLY ADD TO THE BOTTOM OF THE TUBE. This
means picking up the tube and watching the addition!!!!
Cap the strip tubes and place on ice. When the class is all caught up, all strip tubes will be placed in the
thermal cycler (PCR machine) and run through the following program:
Denaturing: 92°C for 2 minutes , then 30 seconds
Primer Annealing: 50°C for 15 seconds
Extending: 70°C for 15 seconds
Repeat 2-4 for a total of 30 cycles
Supply Ordering and Price Information
Item
ProMega Wizard MidiPrep Kit
Sequencing Kit
Strip Tubes
Strip Caps
TEMED Catalyst
Long Ranger Acrylamide
APS (Solid) Catalyst
Urea (Solid)
10X TBE
Primer(M13F/M13R)
Gel-Loading tips
Source/Catalog
Promega / A7640
Epicentre/SE9101LC
Fisher/054072A
Fisher/054074A
Fisher/BP150-20
Fisher/BMA50611
Fisher/BP17925
Fisher/BP169500
Fisher/ BP1333-1
Licor(829-05565/ 829-05566)
Fisher/21-402-177
Units/Cost
25 rxns for $127.68
1 for $225
125X8 for $115.00
300X8 for $100.00
26 ml for $25
250 for $160
25 g for $16
500 g for $35
1 L for $35
1nmole each
4boxes/262.00