MBB 308: MOLECULAR BIOLOGY AND BIOCHEMISTRY LABORATORY I

SUMMER 2011, WEEK 4

NEXT QUIZ: WEEK 5 LAB BOOKS HAND IN: WEEK 5

Reading for this week:

Read the Week 4 lab manual section. Read Chapter I of text:

Endonucleases: Class I, II and III restriction enzymes (focus here is on Class II section 1.2.1). Exonucleases (section 1.3.3) DNA & RNA Dependent DNA polymerases (section 1.3) Plasmid purification (Section 1.6 & Birnboim NAR PDF) PCR: Read Chapter 2, focus on Taq for now.

Week 4: Reading

Elemental operations

Focus on: Pipetting (small volumes this week) and Calculations! DNA (RNA) quantification Bacterial liquid cultures Plasmid purification (2 plasmids this week) PCR Restriction digests with Class II endonucleases Demonstrate that a plasmid and a PCR product from a plasmid contain the same restriction sites (pCR2.1-TOPO_6S). Construct a plasmid map (pUC19_Histone).

Basic procedures

Protocols

Experiments

Course Objectives: Week 4

UV absorbance: DNA spectra (see any problems with the above spectra?)

UV absorbance: RNA spectra

UV absorbance and DNA/RNA purity (see Web_CT PDF)

RNA: A260/A280 ~ 2.1 to 2.2

DNA: A260/A280 ~ 1.8 Values are pH sensitive

UV absorbance and DNA/RNA purity

Avoid splashes / aerosols (pipette slowly!) Bacteria settle make sure they are suspended before pipetting. Cleanup with bleach. Tube Waste into biohazard containers for autoclaving.

Bacterial liquid cultures

Bacterial Plasmid purification

As discussed last lecture and in the lab manual there are a range of protocols for extracting nucleic acid from organisms.

We have learned how to purify genomic DNA and RNA using silica columns (largely for safety reasons). The plasmid prep you will use this week is very similar to the preps in Week 3, but has been modified so as to retain Covalently Closed Circular (CCC) DNA (i.e. plasmids) using a slight modification of Birnboim and Doly 1979 (see Web_CT).

Cells are rapidly lysed and genomic DNA irreversibly denatured using NaOH. Upon neutralization (in high salt conditions required for silica column binding) the genomic DNA precipitates and is removed by centrifugation. The plasmid DNA (now in the supernatant together with cellular debris) is then bound to a silica column, washed and then eluted as you have done previously using a low salt buffer (water in your case).

Bacterial Plasmid purification

For your first PCR you will be amplifying a region of the pCR2.1-TOPO_6S plasmid directly from a bacterial colony. The colony serves to isolate a particular plasmid. This can be very important if you have a library of DNA inserts for example. The themocycling required for PCR serves to break open the bacteria, exposing the plasmid within as a template for PCR.

Colony PCR

17.1: 5-CAG GAA ACA GCT ATG AC 17.2: 5-GTT TTC CCA GTC ACG AC (+ overhead material) Calculate:

1. Total PCR product size expected in this weeks lab (show your TA!).
2. Size of restriction fragments produced by digestion with EcoRI (show your TA!).

See page 33 of manual, Lecture 2 slide 13-15

Palindromic sequences (Class II), what is the rule here?

5-CCGG GGCC-5 5-AACGTT 5-GCATATGC TTGCAA-5 CGTATACG

5-AT TA-5

Nearly palindromic sequences (Also Class II generalize the above rule?):

5-GCCNNNNNGGC CGGNNNNNCCG-5

5-AACNNNGTT TTGNNNCAA-5

Nonpalindromic sequences (not class II):

5-A T-5 5-AG TC-5 5-ACT TGA-5 5-AGGT TCCA-5

Class II restriction sites

EcoRI: XhoI:

5-G^AATT C C TTAA^G-5 5-C^TCGA G G AGCT^C-5

SstI BamHI PstI Hind III

(Check out the NEB website for last 4, which can be used to perform simultaneous double digests?)

Class II restriction Enzymes in Week 4

Example: SgrA I 5-CR^CCGG YG

GY GGCC^RC-5

Class II binding and cleaving as dimers

2 kb A C

Digestions A B C A&B A&C B&C

Bands Observed 6, 1.5 7.5 7.5 6, 1, 0.5 4, 2, 1.5 4.5, 3

1 kb

Model Plasmid example

B 0.5 kb A A 1 kb 3 kb B 2 kb A 2 kb 4 kb

A B A&B

5, 2, 1 4, 4* 3, 2, 2*, 1

Restriction maps: Building the most reasonable map (linear verses circular maps)

Practice calculations (stay if you like)