Today we lysed our bacteria open, extracted all the molecules inside them, including all their proteins, and then purified our protein of interest, the GFP.
We performed a protein chromatography using Bio-Rad's disposable columns. Our task was separating the GFP from the thousands of other proteins that we would find in a bacterium. The resin used in these columns were chosen to separate specifically the GFP by Hydrophobic Interaction Chromatography (HIC).
In HIC the resin is very hydrophobic. The salty buffer used causes proteins to change their 3-D conformation exposing the hydrophobic amino acids, wich stick to the hydrophobic resin. The GFP sticks especially strongly to the resin. Then we did washes with solutions of decreasing salinity so other proteins that happened to be less hydrophobic than the GFP recovered their original conformations, un-bound from the resin and were washed out. At the end we used a low salinity solution that allowed thr GFP to recover its original 3-D conformation making its hydrophobic amino acids un-available and separating from the resin.
Following this protocol we purified the GFP exclusively, and that's why our little tubes glowed when exposed to UV light.
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Friday, October 10, 2008
Thursday, October 9, 2008
Miscellaneous news and notes
Somebody asked me about the questions for lab 4/5. THat lab was due last week, but here's a summary of the questions that I want for the last few labs:
Lab 4/5: All five questions on page 56 of that lab maual
Lab 6: Questions 1, 4, and 5 on pages 43 and 44 of that manual. Questions on page 54 don't have to be answered in the questions section, but if you can answer them for your self you an write a solid discussion. Use those questions as suggestions to how to write the discussion.
Lab 7/8: Questions on page 38 of that manual. Some people answered more than those, and that will not hurt. It will only help!
Lab 9/10:
Band sizes in the ladder of the GMO lab (lab 6)
Lab 4/5: All five questions on page 56 of that lab maual
Lab 6: Questions 1, 4, and 5 on pages 43 and 44 of that manual. Questions on page 54 don't have to be answered in the questions section, but if you can answer them for your self you an write a solid discussion. Use those questions as suggestions to how to write the discussion.
Lab 7/8: Questions on page 38 of that manual. Some people answered more than those, and that will not hurt. It will only help!
Lab 9/10:
- In your own words, describe cloning
- What is a bacterial colony?
- In your won words describe hydrophobic interaction chromatography (HIC) and identify its purpose in this lab
Band sizes in the ladder of the GMO lab (lab 6)
- 1000 bp
- 700 bp
- 500 bp
- 200 bp
- 100 bp
LAB - GFP purification through chromatography
Today we started the process to purify the Green Fluorescent Protein (GFP) from the bacteria we transfromed last week.
The process was really short: Just picking a colony from each of the LB/amp/ara and LB/amp plates, usit to inoculate a tube with a few mLs of LB/amp/ara broth, and sticking the tubes in a shaker/incubator at 32ºC and an undisclosed number of RPMs. Undisclosed, because the knob is not in RPMs, but through experience I would estimate that we set it at about 200 rmps.
The tubes were set so they are in a diagonal position, so the surface area of the medium is larger than in vertical position, promoting a better uptake of O2 from the air, thus enhancing the efficiency of bacterial growth.
Tomorrow morning we will do the actual chromatography to isolate the GFP.
-----------------------------------
The process was really short: Just picking a colony from each of the LB/amp/ara and LB/amp plates, usit to inoculate a tube with a few mLs of LB/amp/ara broth, and sticking the tubes in a shaker/incubator at 32ºC and an undisclosed number of RPMs. Undisclosed, because the knob is not in RPMs, but through experience I would estimate that we set it at about 200 rmps.
The tubes were set so they are in a diagonal position, so the surface area of the medium is larger than in vertical position, promoting a better uptake of O2 from the air, thus enhancing the efficiency of bacterial growth.
Tomorrow morning we will do the actual chromatography to isolate the GFP.
-----------------------------------
Tuesday, October 7, 2008
Chromosomes
Yesterday and today we covered pages 202-219 and 233-245 in chapter 4. We talked mostly about chromosomes, how they are composed, what are their main features, how they are studied and how they are used to study the genomes of some organisms.
Reading for next week: pages 245-260
Quiz # 6 Q&As:
1. What are the three main components of a nucleotide?
Nitrogenous base, phosphate group, sugar
2. Why are the ends of a DNA strand called 5’ and 3’?
Carbons 5' and 3' in the sugar of a nucleotide are the ones that form covalent bonds with other nucleotides. At the ends of a DNA strand a 5' carbon (more exactly the phosphate group associated to it) and a 3' carbon will be open, and potentially ready to bind to another nucleotide.
3. What does it mean that DNA strands are antiparallel?
The 5' end of a strand matches up with the 3' en of the other
4. What does it mean that DNA strands are complementary?
Because the complementarity of the nitrogenous bases once you have the sequence of one strand of DNA you can infer the sequence of the complement.
The association between DNA and some proteins (mainly histones). They very basis of chromosomal structure and function.
---------------------
Quiz # 7 Q&As:
1. What are homologous chromosomes?
In diploid organisms they are pairs of chromosomes that have the same genes. One of the chromosomes is inherited from the father, the other from the mother.
2. What is a karyotype?
The organized set of chromosomes of a given organism. They are stained and homologous chromosomes paired so they can be studied.
3. What is a transposon?
A fragment of DNA that changes locations in a genome, or that it replicates it self so the copy can insert itself somewhere else in the genome.
4. Mention three kinds of transposons and explain one
LINEs, SINEs, repetitive sequences (miscrosatellites, minisatellites), retroviral - like sequences (explain either one of them)
5. What is the name of the histone complexes around which DNA wraps in the chromosomes?
Nucleosomes
----------------------------
Reading for next week: pages 245-260
Quiz # 6 Q&As:
1. What are the three main components of a nucleotide?
Nitrogenous base, phosphate group, sugar
2. Why are the ends of a DNA strand called 5’ and 3’?
Carbons 5' and 3' in the sugar of a nucleotide are the ones that form covalent bonds with other nucleotides. At the ends of a DNA strand a 5' carbon (more exactly the phosphate group associated to it) and a 3' carbon will be open, and potentially ready to bind to another nucleotide.
3. What does it mean that DNA strands are antiparallel?
The 5' end of a strand matches up with the 3' en of the other
4. What does it mean that DNA strands are complementary?
Because the complementarity of the nitrogenous bases once you have the sequence of one strand of DNA you can infer the sequence of the complement.
The association between DNA and some proteins (mainly histones). They very basis of chromosomal structure and function.
---------------------
Quiz # 7 Q&As:
1. What are homologous chromosomes?
In diploid organisms they are pairs of chromosomes that have the same genes. One of the chromosomes is inherited from the father, the other from the mother.
2. What is a karyotype?
The organized set of chromosomes of a given organism. They are stained and homologous chromosomes paired so they can be studied.
3. What is a transposon?
A fragment of DNA that changes locations in a genome, or that it replicates it self so the copy can insert itself somewhere else in the genome.
4. Mention three kinds of transposons and explain one
LINEs, SINEs, repetitive sequences (miscrosatellites, minisatellites), retroviral - like sequences (explain either one of them)
5. What is the name of the histone complexes around which DNA wraps in the chromosomes?
Nucleosomes
----------------------------
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