IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/06/17
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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.
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.
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...
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:
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 ✓ and Agarose gel ✓ to confirm PCR and PCR purification.
Restriction Digest and Agarose Gel
Restriction Digest reactions were set up as follows:
NOSterm and NOSterm + STOP:
BioBrick Plasmid V0120 (Ampicillin Resistance):
Reactions were allowed to proceed for 45 minutes at 37°C.
Following QIAgen Plasmid DNA Purification Protocol Using the QIAprep Spin Miniprep Kit and a Microcentrifuge the following plasmid was purified:
Nanodrop Values for VP16
Quantities per double digestion reaction
Single digest reaction
ingredients combined and all tubes were put in 37°C bath for 10 minutes
Digest with SPE1 and XBA1
Gel Recipe: 1 X TAE
Nanodrop values for resulting DNA
Tube placed in -20 freezer labeled VP1 fragment XBA SPE1
Bacterial transformation of GAL4 DNA binding domain
BBa_K105007, plate 3 well 9I, psB1A2