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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

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