Lecture 3 Flashcards
Why do we isolate mRNA
cDNA library construction
Use cDNA as a template for pcr amplification
Why do we use gel electrophoresis for
Common technique
Used to analyse a sample of DNA and RNA and check size and quantity of DNA fragments
What can gel electrophoresis confirm
Presence of DNA or RNA in a sample
Seperate different sized molecules
Determine the size of DNA and RNA molecules
What are the gels made of
Agarose
When elextrix is applied to the gels what happens to the DNA and RNA molecules
Migrate towards the positive electrode
How does the gel know the size of DNA and RNA
It acts like a sieve the smallest fragments will migrate faster than the larger fragments
Treating the gel with a stain will do what
Allow fragments of DNA and RNA to be seen under uv light
What will migrate faster supercoiled or linear DNA
Super coiled
Name the 6 steps in making a cDNA library
- Extract RNA from a tissue that expressed the gene
- Purify mRNA
- Using reverse transcriptase convert mRNA into double stranded mRNA cDNA hybrids
- Replace the mRNA strand of the cDNA mRNA with DNA
- Add sticky ends to the double stranded cDNA
- Ligase the cDNA into a suitable vector to generate the cDNA library
What is used to build the 2nd strand cDNA
Use RNAse H and DNA polymerase I
Total RNA composition
80% ribosomal RNA
15% transfer RNA
2-5% messenger RNA
What do eukaryotic mRNA have at the end
Poly A tail
Do rRNA and tRNA have a poly A tail
No
How are sticky ends added to the cDNA
Ligase it with adaptors
cDNA library is used fo
Fictional complementation
Screening by homology
Pcr template
What are sequence specific endonucleases
Restriction enzymes produced by some bacteria
How many different restriction enzymes are there
3000
What do type 2 restriction enzymes do
Recognise and cleave at specific DNA sequences
How are DNA fragments produced
Restriction enzyme digestion
What is an isoschizomers
Different restriction enzymes that recognise the same sequence
What are neoschizomers
Different restriction enzymes that recognise the same sequence but cut it differently
How could we get sticky ends
When different enzymes with different recognition sequences
What bond is involved with DNA Ligase
Covalent bonding in the DNA fragments
What temp can Ligase reactions work
25C
16C
4C
How are DNA and RNA extracted
By lysing cells, denaturing, phenol removal of protein and ethanol precipitation of nucleic acid
Why do we isolate DNA
For gdn library construction
Use as a template for pcr amplification
