GenLabFinal
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- First 6 Steps of Cloning
- 1.Isolate mtDNA from cheek cells-Isolate DNA 2.PCR-Cut 440bp(Seq. wanted) & PCR amplifies seq. 3.Gel Electrophoresis-Screening to verify 440bp 4.Ligate mtDNA into plasmid-For Cloning 5.Transformed into E.Coli(competent)-amplify(2^n) & used for screening 6.TET screening-only bacteria w/ insert grows on TET plate
- Lab 6 Name and Purposes
- Bacterial Transformation and Cloning -Insert ligated into plasmid -Transformed into E.Coli for cloning(mtDNA if PCR worked, if not, then ORF7/ORF58)
- E.Coli facts
- -replicates quickly -2 defenses-plasma membrane(heated to make porous) and natural RE
- Plasmid WITH inserted DNA on TET plate
- -insert disrupts CI gene -No lambda repressors are formed -RNA Polymerase can proceed -TET Resistance gene is expressed
- Lambda Repressors
- Regulatory proteins -prohibit RNA Poly. from transcribing
- Plasmid WITHOUT inserted DNA on TET plate
- -CI gene is not interupted -Codes for lambda repressors -No RNA Polymerase transcription -No TET Resistance expressed -Forms Control Plate-everything grows
- Kind of Plasmid Used
- GenHunter Plasmid -allows blunt end ligation - complementary ends are not required for ligation so no RE are needed
- Antibiotic used on Transformation plates
- Tetracyclin
- Lab 7 Name and Purposes
- Colony Lysis and PCR -Determine which plate produced most colonies -Why were such colonies present
- How to verify if Plasmid contains Insert after Cloning
- By lysis and then amplifying fragment (PCR)
- Other reasons bacteria grew on TET plate
- False+ = colony grows without insert -Homologous Recessive(ci/ci) -Mutation ---Both alter CI gene
- Colony Lysis Procedure
- Remove plasmids+insert from bacteria -Add Lysis Buffer -Heat cells with buffer -Supernatent-(plasmid+insert)-used for PCR
- Colony Lysate
- Plasmid + Insert aka the Supernatent after heating with Lysis Buffer
- PCR Reagents after Colony Lysis
- -ddH2O -(L)Lgh primer-62bp -(R)Rgh primer-62bp -Colony Lysate(Plasmid+DNAinsert) -Master Mix(buffer,dNTP\'s, and TAQ)
- Reasons for L and R primers for PCR
- Primers are complementary to the plasmid and not the insert -Positive(L) and Negative(R) Primers are used to amplify the inserted fragment
- How to find Expected Base Pair Length of the plasmid for PCR?
- Left primer(62bp) + Right primer(62bp) + Viral/mtDNA length
- DNA Lengths for PCR after Lysis
- mtDNA-440bp ORF7-2087bp ORF58-1073bp
- Lab 8 Name and Purposes
- Gel Electrophoresis and Extraction -Detect DNA that was amplified by PCR -Determine if cloning was successful (check bp length) -SIZE OF PCR PRODUCT DETERMINES WHETHER BACTERIAL CLONE CONTAINS PLASMID ALONE OR PLASMID WITH INSERT
- Reagents used for Lab 8 (Extraction)
- -NT Buffer-solubilizes buffer -NT3 Buffer-removes agarose contaminants -NE Buffer-Elutes(removes) PURE DNA;changes pH
- Ethidium Bromide
- EtBr-used to make Buffer for GE;makes flourescent under UV light by INTERCALATING between DNA
- Gel Ingredients
- Agarose + Buffer
- Buffer Ingredients
- TAE + ddH2O + EtBr
- Reading the Ladder for (Plasmid+Insert)
- -mtDNA-440bp -ORF58-1000bp -ORF7-2000bp(just above the ladder)
- C.U.C and use
- Clean Up Column-to filter buffers
- Primer Dimer
- spot at bottom of ladder
- Band should show on ladder if:
- -Bacterial Clone contained a plasmid with insert -PCR reaction worked
- Lab 9 Name and Purposes
- Restriction Enzyme Digest -Digest bands using two differents RE -Determine which gene we have -If RE cuts at correct spots, then RE Digest worked
- Previously cut plasmid/PCR bands after Lysis
- mtDNA-564bp ORF7-2111bp ORF58-1197bp
- RE Mapping Criteria and Buffers used
- [When using ORF7/ORF58, add 62bp to each cut] -mtDNA-#4 -ORF7-#2+#3 -ORF58-#2+#4
- RFLP and Uses
- Restriction Fragment Length Polymorphism analysis -used to trace inheritence -Presence/Absence of a cut results in GENETIC DIVERSITY
- RE Digest Reagents
- -10X TAE Buffer(flourescent) -Purified PCR product -ddH2O -Specific RE
- Lab 10 Name and Purposes
- Bioninformatic Investigations of Human Genetic Diversity using mtDNA -Different sequences can be compared to identify individuals ancestry
- Use of computer software or databases to analyze RNA/DNA/Protein sequences and to probe the structure and function of the sequences
- Bioinformatics
- PubMed
- maintained by NCBI
- NCBI
- National Center for Biotechnology Information (run by NIH)
- NIH
- National Institute of Health
- BLAST and purpose
- Basic Local Alignment Search Tool -compares sequences to other sequences
- Using computers to investigate biological properties
- In Silico
- Phylogenetic tree that depicts E.D. between sequences
- Cladogram
- Nucleotide Differences
- # of differences between 2 sequences -more differences, more evolutionary difference