Problem shooting
During the experiment, we
found many problems other than experimental errors. We list them here,
hoping to be helpful to the future team.
1. DNA design: When designing primers, we should not only check whether
dimers will be formed between primers, but also check whether primers
themselves will form partial complementarity. This is fatal for the
fluorescent probe, because the local double chain will cause the probe
to be cut in the absence of the template, resulting in serious
false-positive phenomenon.
2. Single strand preparation: There are some tips if you want to use
agarose gel electrophoresis for single strand density identification. On
the one hand, the sensitivity of GelRed to single strands is at most
only half of the double strands. On the other hand, the single chain
usually has its secondary structure, and the rate of single strand DNA
swimming in gel does not depend entirely on molecular weight. Therefore,
test experiments need to be done to verify that the received DNA is
indeed the target single strand.
3. Protein expression: When expressing a protein, you need to find out
whether the protein is a secretory protein or an intracellular protein.
Generally, the proteins expressed in E. coli are intracellular proteins,
which means you have to do cell lysis. If the expression of the target
protein cannot be detected in the supernatant after intracellular
protein lysis, it is likely that the protein is in the inclusion body.
Do not give up and start again directly. If the protein disappears after
passing through the chromatographic column, it may be caused by the
incorrect concentration of the eluent, or you may need to add protease
inhibitors.
4. If the fluorescence signal is strong during fluorescence detection, a
black 96 well plate or 384 well plate shall be used. If you especially
want to use a white board, be careful to avoid interference from
adjacent holes. This can ensure that the data obtained is normal. Even
if you operate very quickly, it is difficult to make each hole react at
the same time (we will describe this in detail in future), so you can
add a hole every 10s and finally translate and align.
5. ssDNA prepared by long and short chain method: If you think the
agarose gel electrophoresis is unreliable, we can also provide another
way. A spacer was added at the 3’end of the antisense chain primer,
followed by 20 nt poly-A. Two single chains can be separated by length
through modified page glue with fluorescent dye. However, this method
takes a long time, and then dialysis is needed to remove the urea used
for denaturation. But the concentration of single chain is higher than
that of agarose gel electrophoresis.
