Download PCR for TOPO Cloning

Probe qPCR Testing Results, PCR for TOPO Cloning, TOPO Cloning
Leaning, Nested PCR for TOPO Cloning
Technical Log
Name: HaoQi (Esther) Li
Date: 7/11/06 Tuesday
Work Location: Naval Medical Research Center, Silver Spring, MD
Title of Project: Developing an Optimized qPCR for Borrelia lonestari
1. Objective – Why did you hope to accomplish today?
I hope to read more about the TOPO PCR Cloning and the Smart Cycler. I will also check the
second Probe qPCR test. If possible, I will do the TOPO plasmid cloning. Perhaps Dr. Ju will
show me how to do the standard test.
Since Dr. Wood and Mr. Pearce will visit today, I hope I’ll get my ISEF forms all fixed.
2. Events – a. summarize the schedule of events. Procedures performed in detail, results,
analysis, with tables, charts, diagrams, and scanned sketches
b. summarize communications, emails (attached), conversations, pertinent material read
(annotated bibliography)+ reactions, browser used (search identifier, annotation)
Probe qPCR Testing 2 Results:
This made me so happy:
The assay and DNA samples WORK!!!!!   
I asked Dr. Ju why Sample 1 crossed the threshold earlier than Sample 2; since she prepared
Sample 1 and I prepared Sample 2, I wondered if the reason was whether my biotech
performance was not as good as hers or Sample 1 just had a higher concentration of DNA. Dr.
Ju assumed the latter.
PCR for TOPO Cloning
Since the assay and DNA worked, it was time to do gene cloning. First, we have to do a PCR for
the entire gene, with procedures as shown:
Before I came in the morning, Dr. Ju resuspended and diluted the primers (not the primers in the
assay, but primers for the entire flagellin gene) into 20μl. These primers ordered from MWG,
http://www.mwg-biotech.com/html/all/index.php, which was not the same company as where the
assay was synthesized.
The primers are 11F and 970R, for the whole gene, not just the assay.
Date:
7/11/2006
Final conditions
Vol. Template
Vol. Reaction
Forward Primer
Reverse Primer
Reagent
Water
superMix 1.1X
Forward Primer
Reverse Primer
Specificity:B. lonestari
PCR Stage1:
2 ul
Stage2:
25 ul
0.3 uM
0.3 uM
Stage3:
Stock
22 ul/each
20 uM
20 uM
Total(Before Template Addition)
Each(Before Template Addition)
No.Samples 1
hold 94C: 2min.
3-Temp 40 cycles
94C: 30sec.
55C: 30sec
72C: 1min.
hold 72C: 7min.
Master Mix
0.24 ul
22 ul
0.38 ul
0.38 ul
23 ul
23 ul
The red Biometra Tgradient PCR machine was used instead of the usual white PE Applied
Biosystems GeneAmp PCR System 9700 machine, because Joey was using the latter.
(Dr. Chan talked to me about the dirty hood (Bio Safety Cabinet) filter cleaning tomorrow. So
during 1630 hours to 0800 the next day, no one should be in the lab which will contain high
concentrations of carcinogen formaldehyde. Dr. Chan said that paraformaldehyde will be boiled
into formaldehyde for two hours. Then after the two hours, ammonium bicarbonate will be used
to neutralize the toxic gas.)
TOPO PCR Cloning Leaning:
http://www.invitrogen.com/content/sfs/manuals/topotaseq_man.pdf
Purify PCR product using Qiagn Purification Kit
I wrote down the procedures on a piece of paper. Then I did some research about competent
cells and heat shock: http://en.wikipedia.org/wiki/Competent_cell So competent cells are
bacteria that are able to accept vector plasmid.
(Then Dr. Richards had a meeting with us and I told everybody the good news of my functioning
assay, which I also explained two hours later to Dr. Wood and Mr. Pearce. Mr. Pearce checked
all my ISEF forms.)
PCR Results:
Then I prepared a gel and loaded in the middle with Joey’s sample on one side and the 100bp
standard on the other. Unfortunately, none of our samples showed up, the red oval below
indicates the position of around 1000 bp where the band should have been. So Dr. Ju said that
that might have been caused by the low concentrations of DNA in my sample, so I would do a
nested PCR.
Nested PCR For TOPO Cloning:
Here is a diagram that might help explaining why I did three samples for the nested PCR:
B. lonestari flagellin gene:
: region of base pair 11 Forward Primer (primer for the whole gene)
: region of base pair 594 Forward Primer (primer inside the assay, for the probe)
: region of base pair 655 Probe (unique to B. lonestairi inside the assay)
: region of base pair 719 Reverse Primer (primer inside the assay, for the probe)
: region of base pair 970 Reverse Primer (primer for the whole gene)
So the nested PCR instead of using the 11Forward Primer and the 970Reverse Primer like the
first set of PCR, I had three samples. A = Assay.
Since 11 F and 970 R have 20μl concentration, but 594F and 719R have 10μl concentration,
the amount of 594F and 719R needs to be doubled and no water is added for tubes 2 and 3.
Then the amount of 2 μl template DNA was the product of the first PCR. Everything else is
the same as the PCR above.
1
2
3
AA with 594 F + 719 R
FA with 11 F + 719 R
AR with 594 F + 970 R
0.38 μl for both primers
0.76 μl for F, 0.38 μl for R
0.38 μl for F, 0.76 μl for R
0.24 μl of H2O
0 H2O
0 H2O
After I set the nested PCR running, I went home.
3. Reflections:
a. actually accomplished: I reviewed the results of yesterday's qPCR run, and then I started
PCR amplification for TOPO Cloning, but it didn't show up well, so I started the nested PCR
process. I explained my project in person to Dr. Wood and Mr. Pearce with Dr. Ju and Dr.
Richards also listening. I was encouraged by their constant smiles.
b. concerns: I hope that the Science Fair Committee will agree with Dr. Richards that I do not
work with any potentially hazardous materials.
c. learning (workplace, science, project, yourself): I learned about the TOPO Cloning
procedures.
4. Planning
When I get home, I will first send Mr. Pearce the name of the bacteria I’m working with, which
is B. lonestari, and then I will review my DNA I notes about bacterial transformation and review
the mechanics behind the heat shock.