IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/06/17

{| width="800"
 * style="background-color: #EEE"|[[Image:igem-logo-150px.png|150px]] iGEM Project name 1
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

Procedures
Gel Electrophoresis

Today, we loaded gel slots to run our 6 DNA samples against a 1kb ladder. The order of our slots were: ladder, ladder, sense 1, sense 1, sense 2, sense 2, sense 3, sense 3, antisense 1, antisense 1, antisense 2, antisense 2, antisense3, and antisense 3. We had to load two slots because there was more than 50 microliters of DNA and it would not fit into one slot. Since we are hoping to collect and purify the DNA after the electrophoresis, we want to run as much of the sample as possible. We also loaded two ladders. We ran the gel for 30 minutes at 100 volts. Our goal is to have the majority of the amplified DNA to be 300bps long.

Thirty minutes later, we examined our gel under UV light. All of the DNA were smaller than 100bp, suggesting that the Phusion Polymerase did not amplify the correct DNA segment. Our sample looks like a primer dimer.

Results
If our Phusion Polymerase had worked, then we would have continued to extract the correctly formed DNA segments, performed a restriction digest and cut the PCR products into a B0120 biobrick with Xba1 and Pst1, ligate, and transform into E. coli. However, since we do not have the correct DNA, we cannot move forward.

Errors

There are several reasons for why our gel did not work. The most likely reasons is that the RNA extraction did not work properly. We used strawberry fruit as our sample for RNA extraction. Because RNA is constantly being degraded by RNAases, the integrity of our RNA may have been compromised before the addition of the denaturant.We will re-extract DNA from our plant samples, now including arabidopsis, probably tomorrow.

Another potential error could have occurred during the Phusion Polymerase. We set our annealing temperature at 72°C, which could have been too high for three of our four primers. We'll rerun our PCR with a gradient PCR of annealing temperatures to see if we can get a better result. The amount of DNA added by template was also much higher than the optimal 50~250 nanograms per 50 micrograms of PCR reactants. We will dilute the DNA templates to 100 micromolar and use one microliter per reaction so that there are approximately 100 nanograms of DNA per reaction.

The Next Step...
Corrections

Redo gel electrophoresis with all three concentrations of Antisense and Sense, each at a range of temperatures, from one to three degrees Celsius above the lower annealing temperature of the primers. This will give three tubes of each of three concentrations of both Sense and Antisense, for a total of 18 tubes. Run gel for 30 minutes at 100 volts with a 100kb ladder.

Corrections were unsuccessful. We are going to go further back and do more research on the strawberry allergen and see if we can obtain a better RNA and DNA sample.

Strawberries are also non-climacteric fruits, which means that they cease to respire after being picked. Our sample strawberries were picked from a grocery store, so whatever mRNA responsible for ripening would have degraded after picking. Climactic fruit, like tomatoes and apples, continue to metabolize sugars and ripen after being picked. The mRNA we were hoping to obtain is related to the ripening process of strawberries, but since our strawberries were not fresh, it is possible that all of the mRNA responsible for coding Fraa1 had degraded before our RNA extraction was performed.

Further procedures If the gel electrophoresis is successful and the pieces are 300bps, then we will perform:


 * Gel Extraction - remove the correct Antisense and Sense DNA segments
 * Restriction Digest - cut the DNA segments with Eco1 and Xba1 (keeping A and S separate)
 * Ligation - links sense, intron, and antisense into a V0120 plasmid (Biobrick)
 * E. coli Transformation - insert the plasmid into bacteria

PCR Purification of pORE Vector Parts
Following QIAgen PCR Purification Kit Protocol the following PCR products were purified: To do: follow up with restriction digest &#x2713; and Agarose gel &#x2713; to confirm PCR and PCR purification.
 * 1) pENTCUP2 Promoter - 16.1 ng/μL
 * 2) NOSterminator Sequence - 76.3 ng/μL
 * 3) STOP codon + NOSterminator Sequence - 76.5 ng/μL

Restriction Digest and Agarose Gel
Restriction Digest reactions were set up as follows:

pENTCUP2 Promoter:
 * 27μL PCR product
 * 3μL Loading Buffer w/ dye
 * 1μL Xba1 Fast Enzyme
 * 1μL Pst1 Fast Enzyme

NOSterm and NOSterm + STOP:
 * 9μL PCR product
 * 1μL Loading Buffer w/ dye
 * 0.5μL Xba1 Fast Enzyme
 * 0.5μL Pst1 Fast Enzyme

BioBrick Plasmid V0120 (Ampicillin Resistance):
 * 3μL DNA
 * 1μL Loading Buffer w/ dye
 * 6μL diH2O
 * 0.5μL Xba1 Fast Enzyme
 * 0.5μL Pst1 Fast Enzyme

Reactions were allowed to proceed for 45 minutes at 37°C.

VP16 Miniprep
Following QIAgen Plasmid DNA Purification Protocol Using the QIAprep Spin Miniprep Kit and a Microcentrifuge the following plasmid was purified:
 * VP16

Protocol

 * 4ml from each overnight cell culture of VP16 (1,2&3) were transferred into 5 ml conicals and spun for 6 minutes at 4400 rpm
 * remainder of overnight cell cultures were placed in the fridge for transfer to microcentrifuge tubes at a later time
 * excess LP+amp decanted, the pellet resuspended in 250μL P1
 * contents transferred from each 15ml conical to a new epindorf tube
 * 250 μL P2 added to each tube, mixed by inverting gently 4-6 times
 * 350 μL N3 added to each tube, mixed by inverting as before
 * tubes centrifuged for 10 minutes at 13,000 rpm
 * supernatants pipetted into new QIAprep sin columns
 * columns centrifuged for 30-60 seconds, flow through discarded
 * QIAprep columns washed with 0.5 ml buffer PB each and centrifuged for 30-60 seconds
 * columns washed with 0.75 ml buffer PE and again centrifuged for 30-60 seconds
 * flow through was discarded and tubes centrifuged for an additional minute
 * column was placed in a clean 1.5 ml microcentrifuge tube
 * 50 μL buffer EB was added, tubes were let stand for 1 minute and centrifuged for one minute after
 * flow through kept and purity tested

Nanodrop Values for VP16

 * VP16 #1
 * 95.7 ng/μL
 * 260/280 = 1.92


 * VP16 #2
 * 102.4 nm/μL
 * 260/280 = 1.92


 * VP16 #3
 * 90.7 ng/μL
 * 260/280 = 1.92

Digestion
Quantities per double digestion reaction
 * 10μL sample
 * 1μL EcoRI
 * 1μL PSD1
 * 2μL buffer
 * 6μL H20

Single digest reaction
 * 10μL sample
 * 1μL EcoRI
 * 2μL buffer
 * 7μL H20

ingredients combined and all tubes were put in 37°C bath for 10 minutes

Digest with SPE1 and XBA1
 * 10μL sample
 * 1μL SPE1
 * 1μL XBA1
 * 2μL buffer
 * 6μL H20


 * All 3 epindorfs of VP16 were combined for a total volume of 90 μL or 9μg
 * 1 unit enzyme cuts 1 μg DNA in 30-60 minutes

Digestion Gel
Ingredients
 * digested enzymes from above reaction
 * 1 eppendorf containing 2μL uncut VP16 and 18μL H2O
 * kb ladder
 * IMAGE TO BE ADDED + details

Gel Recipe: 1 X TAE
 * 900 ml dH2O
 * 100 ml TAE

1 gel
 * 150 ml buffer TAE
 * add ~2.8g agarose
 * mix in beaker
 * microwave until completely dissolved
 * add 5 μL ethedium bromide per 100 ml gel after solution has cooled (no longer steaming)
 * pour gel and let sit til solidified

Procedure
 * run products on gel for ~45 minutes
 * cut out band containing DNA fragment
 * follow extraction protocol in QIAquick spin handbook for QIAquick gel extraction kit protocol

Nanodrop values for resulting DNA
 * 5.6ng/μL
 * 260/280 = 2.24

Tube placed in -20 freezer labeled VP1 fragment XBA SPE1

Glycerol Stocks of VP16
 * 0.5 ml 80% glycerol stock combined with 0.5 ml overnight culture of colony #2 of V{16 TP{10 cells
 * final product placed in the team fence box after mixing in the -80°C freezer

Bacterial transformation of GAL4 DNA binding domain
BBa_K105007, plate 3 well 9I, psB1A2

Procedure
 * removed 1 tube TOP10 chemically competent E.Coli and placed on ice
 * cleaned biobrick depository w/ ethanol
 * pipetted 1 μL of plasmid into the TOP10 cell tube
 * placed TOP10 cell tube on ice for 30 minutes
 * heat shocked at 42°C for 30 seconds
 * placed on ice for 2 minutes
 * added 170 μL SOC medium
 * streaked on LB + amp paltes, incubated at 37°C overnight


 * }