NEED ASAP Using the list provided (note you may not need all the steps listed) state the steps in cDNA production 1. Confirm size of bands by comparing to molecular size marker. 2. Ligate oligonucleotides onto blunt ended product. 3. Cut the cDNA with a unique restriction enzyme. 4. Ligate the cDNA and vector followed by transformation of E. coli. 5. Plate cells onto LB-ampicillin plates containing X-gal. 6. Choose colonies that are white in colour. 7. Cut plasmid with unique restriction enzymes. 8. The size of the clone is small and it will ensure that the entire gene will be found in one fragment. 9. Employ replica plating technique and select cells that are ampicillin-sensitive and tetracycline resistant. 10. Blot petri plates with a nitrocellulose filter, lyse the cells and denature the DNA with a dilute sodium hydroxide solution. 11. Blot electrophoresis gel with nitrocellulose filter and denature the DNA with a dilute sodium hydroxide solution. 12. Flood the filters with radioactive probe and hybridize to immobilized DNA. 13. Choose colonies that are blue in colour. 14. Wash the filter to remove unhybridized probe and expose to x-ray film. 15. Select colonies that correspond in position to the radioactive loci on the filter. 16. Select bands that correspond in position to the radioactive loci on the filter. 17. Cut recombinant plasmid with unique restriction enzymes. 18. Degrade RNA and add DNA polymerase I, clip hairpin using S1 nuclease. 19. Select cells by spread plating onto LB-ampicillin plates. 20. Run the products on an agarose gel. 21. Isolate RNA from cells and use reverse transcriptase along with dNTPs and oligo-dT primers. 22. Synthesize a probe by incorporating radiolabeled dATP into L gene DNA. 23. Sequence the gene fragment and compare sequence to known sequences using bioinformatics.

Human Heredity: Principles and Issues (MindTap Course List)

11th Edition

ISBN:9781305251052

Author:Michael Cummings

Publisher:Michael Cummings

Chapter13: An Introduction To Genetic Technology

Section: Chapter Questions

Problem 20QP: Analyzing Cloned Sequences A base change (A to T) is the mutational event that created the mutant...

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NEED ASAP Using the list provided (note you may not need all the steps listed) state the steps in cDNA production 1. Confirm size of bands by comparing to molecular size marker. 2. Ligate oligonucleotides onto blunt ended product. 3. Cut the cDNA with a unique restriction enzyme. 4. Ligate the cDNA and vector followed by transformation of E. coli. 5. Plate cells onto LB-ampicillin plates containing X-gal. 6. Choose colonies that are white in colour. 7. Cut plasmid with unique restriction enzymes. 8. The size of the clone is small and it will ensure that the entire gene will be found in one fragment. 9. Employ replica plating technique and select cells that are ampicillin-sensitive and tetracycline resistant. 10. Blot petri plates with a nitrocellulose filter, lyse the cells and denature the DNA with a dilute sodium hydroxide solution. 11. Blot electrophoresis gel with nitrocellulose filter and denature the DNA with a dilute sodium hydroxide solution. 12. Flood the filters with radioactive probe and hybridize to immobilized DNA. 13. Choose colonies that are blue in colour. 14. Wash the filter to remove unhybridized probe and expose to x-ray film. 15. Select colonies that correspond in position to the radioactive loci on the filter. 16. Select bands that correspond in position to the radioactive loci on the filter. 17. Cut recombinant plasmid with unique restriction enzymes. 18. Degrade RNA and add DNA polymerase I, clip hairpin using S1 nuclease. 19. Select cells by spread plating onto LB-ampicillin plates. 20. Run the products on an agarose gel. 21. Isolate RNA from cells and use reverse transcriptase along with dNTPs and oligo-dT primers. 22. Synthesize a probe by incorporating radiolabeled dATP into L gene DNA. 23. Sequence the gene fragment and compare sequence to known sequences using bioinformatics.

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