HWK 9. Model answer: Mix positive control DNA with ligation mix; no or very few transformants

Transcription

1 HWK 9 You are trying to clone a gene form your favorite organism in E. coli using a small plasmid vector. You have obtained no transformants. How would you attempt to solve this problem? Rather than listing lots of things one could do, select what you would do first and explain why and how would do, and how you would interpret the result. Make it short and interesting; try something new! Model answer: As a positive control use a very small amount of undigested E. coli vector to transform the competent cells. If you obtain the expected number of transformants, the cells were normally competent to take up DNA, and the selection medium was correct. If the control failed retry with the control plasmid using fresh competent cells and fresh solutions, if possible borrowed from a successful colleague. You should also have a negative control without plasmid DNA to make sure that the competent cells or some solution was not contaminated with plasmid DNA. If the positive control worked, but your ligation mix failed Possible reason (advice) 1 Ligation mix contains phenol, SDS, etc. preventing transformation (purify ligated DNA before transformation) 2 Ligation did not work efficiently (choose correct ratio and concentration of DNA) 3 Insert UV damaged during gel purification (avoid UV) 4 DNA ends incorrect (not blunt, not complementary, not digested, both without 5 P) 5 Cloned sequence lethal for E. coli host (unusual codon usage, inverted repeats) 6 Host-specific restriction (try transforming an non-restricting E. coli mutant strain (e.g. DH10B) How to find out; result Mix positive control DNA with ligation mix; no or very few transformants Check on gel; no difference before and after ligation or too much ligation Did you use 254 nm UV or excessive 360 nm UV? If possible check bands on agarose gel. PCR products are not 5 phosphorylated, extra A may have been added Usually a few transformants containing mutant plasmids will be observed. Plasmids from these will give high transformation frequencies Some E. coli strains destroy methylated DNA isolated directly from plants or animals.

2 Surprise Test (on paper, only public codes, please do not use the internet) Work in pairs. You will be marking the test yourselves before I collect the papers. I want to find out how much you have learned in the lessons, and what you already know from elsewhere If cannot find an answer, write down questions that might lead you to the answer. General questions G1 Which of these does your teacher think are the two most important qualities scientists should have? Great ideas, read many papers, work hard, be very clever, ask good questions, working efficiently, honesty. G2 Explain to me the Chinese name of one lab equipment of your choice. (E.g. centrifuge, pipette, water bath, etc.) Writing a scientific paper W1 W2 W3 Which part of the manuscript should you prepare first. How should it be done? What comes last in the preparation of a manuscript? How much time should you reserve for writing? Agarose gel-electrophoresis A1 Explain how you can change agarose gel conditions to slow down circular DNA more than linear DNA (this can be useful if you need to purify linear DNA fragments and make sure that there are no circular plasmids in your sample. Circular plasmids transform very efficiently and may ruin your cloning experiment.) A2 You separate undigested plasmid DNA on a gel. How can you find out whether a particular band represents a monomeric or dimeric plasmid? (Assume that you know the sequence of the plasmid, but you have no CCC plasmid size standards.) A3 Are the DNA bands in lanes 1 and 4 exactly the same or do the fragments have slightly different sizes? How could you find out without sequencing the fragments? PCR P1 Give three useful controls you should include in your PCR amplification experiments P2 You try to PCR amplify a gene from a new bacterium where you do not know the DNA sequence. All you know is the DNA sequence of an orthologous (similar and same function) gene from a related organism. After PCR amplification using degenerate primers, you obtain a DNA smear. How would you proceed to obtain your desired amplification product? P3 Explain why hot start touchdown PCR helps obtaining the correct DNA fragments. DNA ligation and transformation D1 Electro transformation (electroporation) of E. coli is very efficient. 1 ng (1 nanogram=10-9 g) of 5 kb CCC plasmid DNA may produce more than transformants. How many molecules (order of magnitude is sufficient) produce 1 transformant (bacterium that contains the plasmid)? D2 You have a solution of DNA fragments with complementary sticky ends (e.g. generated by EcoRI). The fragments are 30Kb (1 Kilobases=1000 bp) long. What should you do to obtain 30 kb DNA circles rather than chains of multiple fragments? If you cannot find an answer, write down questions that might lead you to the answer.

3 Surprise Test (on paper, only public codes, please do not use the internet) Work in pairs. You will be marking the test yourselves before I collect the papers. I want to find out how much you have learned in the lessons, and what you already know from elsewhere If cannot find an answer, write down questions that might lead you to the answer. General questions G1 Which of these does your teacher think are the two most important qualities scientists should have? Great ideas, read many papers, work hard, be very clever, ask good questions, working efficiently, honesty. G2 Explain to me the Chinese name of one lab equipment of your choice. (E.g. centrifuge, pipette, water bath, etc.) Writing a scientific paper W1 W2 W3 Which part of the manuscript should you prepare first. How should it be done? What comes last in the preparation of a manuscript? How much time should you reserve for writing? Agarose gel-electrophoresis A1 Explain how you can change agarose gel conditions to slow down circular DNA more than linear DNA (this can be useful if you need to purify linear DNA fragments and make sure that there are no circular plasmids in your sample. Circular plasmids transform very efficiently and may ruin your cloning experiment.) A2 You separate undigested plasmid DNA on a gel. How can you find out whether a particular band represents a monomeric or dimeric plasmid? (Assume that you know the sequence of the plasmid, but you have no CCC plasmid size standards.) A3 Are the DNA bands in lanes 1 and 4 exactly the same or do the fragments have slightly different sizes? How could you find out without sequencing the fragments? PCR P1 Give three useful controls you should include in your PCR amplification experiments P2 You try to PCR amplify a gene from a new bacterium where you do not know the DNA sequence. All you know is the DNA sequence of an orthologous (similar and same function) gene from a related organism. After PCR amplification using degenerate primers, you obtain a DNA smear. How would you proceed to obtain your desired amplification product? P3 Explain why hot start touchdown PCR helps obtaining the correct DNA fragments. DNA ligation and transformation D1 Electro transformation (electroporation) of E. coli is very efficient. 1 ng (1 nanogram=10-9 g) of 5 kb CCC plasmid DNA may produce more than transformants. How many molecules (order of magnitude is sufficient) produce 1 transformant (bacterium that contains the plasmid)? D2 You have a solution of DNA fragments with complementary sticky ends (e.g. generated by EcoRI). The fragments are 30Kb (1 Kilobases=1000 bp) long. What should you do to obtain 30 kb DNA circles rather than chains of multiple fragments? If you cannot find an answer, write down questions that might lead you to the answer.

4 Model answers for Surprise test General questions G1 Which of these does your teacher think are the two most important qualities scientists should have? Great ideas, read many papers, work hard, be very clever, ask good questions, working efficiently, honesty. G2 Explain to me the Chinese name of one lab equipment of your choice. (E.g. centrifuge, pipette, water bath, etc.)

5 The very basics of science Slide No 7 Scientists must be teachers; without teaching knowledge is lost Teachers must explain, and make it interesting so that the pupils want to learn more Scientists must test whether hypotheses (ideas) are correct Great ideas become science only when they are tested experimentally Sadly, many people in the West,including science graduates, fail to understand what scientific testing means, or they dismiss science because it is sometimes wrong They accept the first explanation of a result that comes to their mind They ignore scientific evidence (e.g. smoking) They trust medicines that have not been adequately tested (e.g. thalidomide) (There are many things in life science cannot explain, or which science has not yet tested)

6 Slide No 8 Thalidomide caused this birth defect T. is teratogenic, not mutagenic

7 Writing a scientific paper W1 Which part of the manuscript should you prepare first. How should it be done? Figures and tables. As few lines and colors as possible; comprehensive caption W2 What comes last in the preparation of a manuscript? Polishing and following all the rules of the journal (authors instructions) W3 How much time should you reserve for writing? 30% of the total, about 1 year out of 3

8 Agarose gel-electrophoresis A1 Explain how you can change agarose gel conditions to slow down circular DNA more than linear DNA (this can be useful if you need to purify linear DNA fragments and make sure that there are no circular plasmids in your sample. Circular plasmids transform very efficiently and may ruin your cloning experiment.) Increase agarose concentration (e.g. 0.7% 1.2%) A2 You separate undigested plasmid DNA on a gel. How can you find out whether a particular band represents a monomeric or dimeric plasmid? (Assume that you know the sequence of the plasmid, but you have no CCC plasmid size standards.) Make a partial digest (use a RE that cuts only once) and measure the length of the largest linear band A3 Are the DNA bands in lanes 1 and 4 exactly the same or do the fragments have slightly different sizes? How could you find out without sequencing the fragments? Make dilution to obtain fainter, sharper bands, and run the samples individually and as a mixture. Very small differences in the migration will show up on the gel.

9 PCR P1 Give three useful controls you should include in your PCR amplification experiments No template DNA Primer 1 alone Primer 2 alone P2 You try to PCR amplify a gene from a new bacterium where you do not know the DNA sequence. All you know is the DNA sequence of an orthologous (similar and same function) gene from a related organism. After PCR amplification using degenerate primers, you obtain a DNA smear. How would you proceed to obtain your desired amplification product? Hotstart touchdown; reamplify DNA of approximate size from agarose gel using nested primer(s) P3 Explain why hot start touchdown PCR helps obtaining the correct DNA fragments. Hotstart prevents DNA synthesis (primer elongation) of mis-annealed DNA during the initial warm up Touchdown PCR gives the best chance to the strongest, usually correct, primer-template hybrids DNA ligation and transformation D1 Electro transformation (electroporation) of E. coli is very efficient. 1 ng (1 nanogram=10-9 g) of 5 kb CCC plasmid DNA may produce more than transformants. How many molecules (order of magnitude is sufficient) produce 1 transformant (bacterium that contains the plasmid)? 1 µg 5 kb plasmid molecules; 1 ng 10 9 molecules; Impossible, more transformants than plasmids! D2 You have a solution of DNA fragments with complementary sticky ends (e.g. generated by EcoRI). The fragments are 30Kb (1 Kilobases=1000 bp) long. What should you do to obtain 30 kb DNA circles rather than chains of multiple fragments? 30 kb DNA is about 10µm long. The ends of one linear DNA molecule tend to be very far apart from each other. Ligate at the highest practical dilution