State two purposes of loading dye
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One purpose of loading dye is that it is dense so it weighs down the DNA
sample so that it sinks to the bottoms of the wells and doesn't float in the
buffer solution. A second purpose of loading dye is that it is visible to the
naked eye and moves faster than the DNA, so the loading front serves as
an indicator as of when to turn off the electrophoresis chamber
What happens to the bacteria and DNA when they are first combined and incubated
on ice
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, When they are first combined, the calcium chloride causes the DNA to bind
to the bacteria cell wall and the DMSO helps to assist the plasma DNA to
pass through the lipid cell membrane
Why are the cells grown in C medium?
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It contains a nutrient medium that helps the bacteria enter the growth
phase efficiently. It also has an antibiotic for which resistance is carried by
the plasmid
What sequences will overlap with which primers?
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The pJET SEQ F and pJET SEQ R sequencing primers are designed to
anneal to the pJET 1.2 plasmid outside the MCS. The GAP SEQ F and GAP
SEQ R primers are designed to anneal to the plant GAPC genes
What was the purpose of adding ethanol to the supernatant before you loaded it
onto the column?
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, The purpose of adding ethanol to the supernatant before it was loaded
onto the column was to help DNA bind to the silica column later during the
column chromatography
Why did you run two identical PCR samples containing Jacaranda DNA template?
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We ran two identical PCR samples containing Jacaranda DNA so that if
something, such as contamination or loss of sample, we would have
another one as a backup
What is the importance of growing bacteria on plates containing Amp and IPTG?
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The importance of growing bacteria on plates containing AMP and IPTG is
that the ampicillin kills the bacteria without a plasmid and the IPTG is
artificail lactose, which induces the transcription of the lac operon and
increases the expression of the amp gene so that we can screen out the
bacteria without a plasmid
Where is the DNA after elution--left on the column or in the new collection tube?
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, After elution, the DNA is no longer bound to the column, but is in the new
collection tube. The hot sterile water solubilizes the DNA and allows it to
flow through the column through centrifugation
What would happen to the cloning reaction if the proofreading reaction was done
after the ligation reaction?
Give this one a try later!
If you did the ligation reaction first and then did the proofreading reaction,
you would be attempting to ligate the purified DNA when it still had the 3'
A overhang from the Taq DNA polymerase because it hasn't gone through
the proofreading enzyme yet to get rid of the overhang. Because this DNA
is a sticky end strand, this ligation wouldn't work since we are doing blunt
end ligation using the pJet blunted vectro, which ligates blunt ends that are
all compatible with each other. You would need a more specialized vector
that was cut with the same enzyme as the DNa to get the same
complimentary sticky ends
Why did many of you need to add another 50 uL of lysis buffer after adding 700 uL?
Give this one a try later!
We needed to add another 50 uL of lysis buffer to ensure that the plant
tissue was sufficiently ground into a homogenous lysate because the extra
50 uL because it makes it easier to see if there were any clumps of plant
tissue that still needed to be ground. it also caused there to be more
supernatant after the centrifugation, which made it easier to extract without
touching the pellet of cellular debris at the bottom
Give this one a try later!
One purpose of loading dye is that it is dense so it weighs down the DNA
sample so that it sinks to the bottoms of the wells and doesn't float in the
buffer solution. A second purpose of loading dye is that it is visible to the
naked eye and moves faster than the DNA, so the loading front serves as
an indicator as of when to turn off the electrophoresis chamber
What happens to the bacteria and DNA when they are first combined and incubated
on ice
Give this one a try later!
, When they are first combined, the calcium chloride causes the DNA to bind
to the bacteria cell wall and the DMSO helps to assist the plasma DNA to
pass through the lipid cell membrane
Why are the cells grown in C medium?
Give this one a try later!
It contains a nutrient medium that helps the bacteria enter the growth
phase efficiently. It also has an antibiotic for which resistance is carried by
the plasmid
What sequences will overlap with which primers?
Give this one a try later!
The pJET SEQ F and pJET SEQ R sequencing primers are designed to
anneal to the pJET 1.2 plasmid outside the MCS. The GAP SEQ F and GAP
SEQ R primers are designed to anneal to the plant GAPC genes
What was the purpose of adding ethanol to the supernatant before you loaded it
onto the column?
Give this one a try later!
, The purpose of adding ethanol to the supernatant before it was loaded
onto the column was to help DNA bind to the silica column later during the
column chromatography
Why did you run two identical PCR samples containing Jacaranda DNA template?
Give this one a try later!
We ran two identical PCR samples containing Jacaranda DNA so that if
something, such as contamination or loss of sample, we would have
another one as a backup
What is the importance of growing bacteria on plates containing Amp and IPTG?
Give this one a try later!
The importance of growing bacteria on plates containing AMP and IPTG is
that the ampicillin kills the bacteria without a plasmid and the IPTG is
artificail lactose, which induces the transcription of the lac operon and
increases the expression of the amp gene so that we can screen out the
bacteria without a plasmid
Where is the DNA after elution--left on the column or in the new collection tube?
Give this one a try later!
, After elution, the DNA is no longer bound to the column, but is in the new
collection tube. The hot sterile water solubilizes the DNA and allows it to
flow through the column through centrifugation
What would happen to the cloning reaction if the proofreading reaction was done
after the ligation reaction?
Give this one a try later!
If you did the ligation reaction first and then did the proofreading reaction,
you would be attempting to ligate the purified DNA when it still had the 3'
A overhang from the Taq DNA polymerase because it hasn't gone through
the proofreading enzyme yet to get rid of the overhang. Because this DNA
is a sticky end strand, this ligation wouldn't work since we are doing blunt
end ligation using the pJet blunted vectro, which ligates blunt ends that are
all compatible with each other. You would need a more specialized vector
that was cut with the same enzyme as the DNa to get the same
complimentary sticky ends
Why did many of you need to add another 50 uL of lysis buffer after adding 700 uL?
Give this one a try later!
We needed to add another 50 uL of lysis buffer to ensure that the plant
tissue was sufficiently ground into a homogenous lysate because the extra
50 uL because it makes it easier to see if there were any clumps of plant
tissue that still needed to be ground. it also caused there to be more
supernatant after the centrifugation, which made it easier to extract without
touching the pellet of cellular debris at the bottom
