Suggestions & Tools

Preparation of Short Linear DNA

1. In our experiments, we used several short DNA sequences, some of which are different at only one single nucleotide. At the beginning, we tried making point mutations on the plasmid we bought, but the results were not so perfect and the process took too much time. So we tried Overlap PCR by directly synthesizing four overlap primers and going through certain PCR procedure to get the wanted DNA sequences.

2. In order to prepare a large amount of DNA template, we tried retrieving the PCR product in the agarose gel and using it as the template for further PCR. However, since the gel retrieving efficiency is not high enough, plenty of products were lost and our repeating PCR did not bring more DNA product. So we wouldn't recommend repeating PCR many times when preparing DNA template.

Automatic Design of Nucleic Acid Probe or Flexible Linker

In our project, we tried to find the aptazymes with an appropriate cleave ratio through rounds of selection of our randomized library, add two extra sequences on both ends of the aptazyme and use complementary probes in the test step. Through our modeling work, we tried to predict the result of selection and whether our probe-depending design will work considering the two-dimensional structure of the aptazyme. We build a model which can generate random bases in part of a nucleic acid sequence as required, predict the two-dimensional structure of the corresponding single-strand RNA in batches and pick out the wanted structures and sequences. Beyond our project, this tool can be used in a scene where RNA sequences of special secondary structure are wanted and manual design is too complicated, such as designing oligonucleotide probes for molecular hybridization, biological calculation based on toehold-mediated strand displacement(TMSD), functional nucleic acid design and assembly, and any other similar situations.

Trouble Shooting

In our molecular biology experiments, we were meeting new problems all the time and have summarized a few tips for any future iGEM team to refer to.

1. We have tried to get the short linear sequence on a circular plasmid through PCR amplification. It`s difficult to get the target band at first, and bands higher than 2000bp or much larger DNA fragments staying in gel pores were found. It turned out that the high-speed DNA polymerase like KOD, T5, or Rapid Taq can easily anneal through the whole circular plasmid or even twice in one cycle of PCR. An extremely short annealing time may not be a good solution since these polymerases are active enough even in the denaturing step. The normal Taq polymerase is more suitable for short fragments amplification.

2. Sometimes the efficiency of DNA extraction kit wasn`t high enough. We suggest to let the GDP Buffer (the buffer with high salt concentration to transfer the DNA fragments to DNA absorption column) go through the absorption column twice, dry the absorption column for 15min after the last wash step with EtOH, and wait for 5 min more before centrifuging in the elution step to ensure that the DNA has been completely dissolved. In this way we can get the DNA product in higher concentration with less micromolecular impurities.

3. In the process of large-scale preparation of short DNA templates, we expected to directly use the extracted DNA template to amplify itself through PCR. However, we met the trouble that the PCR products all stayed in gel pores when electrophoresing. In our trial to solve the problem, we found that when diluting the DNA template (about 18ng/μL) 100, 1000 or 10000 times, the product ran out of the gel pores but presented no specific bands; when diluting 100000 or 1000000 times, the DNA product moved as expected and showed specific bright band at the right length in the electropherogram.

4. In T7 transcription reaction, NTP is a significant substrate that provide the monomers of transcription product; but it is also reported that higher NTP concentration can chelate most of the free Mg 2+ in T7 transcription reaction mix. In our experiments, we observed an obvious decrease of transcription efficiency when NTP concentration is higher than 4mM each. The 2mM NTP concentration can lead to the best performance of in vitro T7 transcription reaction.


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