PCR Tricks: How to solve the PCR contamination problem?

In a transgenic research, PCR confirmation of the transgenic events is a must to screen them from the non-transgenic events. The positive control we keep is generally the isolated engineered vector plasmid containing the transgene. A common problem that occurs with most of the beginners in the lab is after keeping a PCR they get positive in all the samples along with positive, negative and blank. This mostly happens due to handling error i.e. In anyway you have ended up adulterating any one or more than one of your components with the template from the positive control vial or from any positive sample. This mostly goes undetected while keeping a PCR reaction and additionally a PCR reaction is really very sensitive.The best method to avoid this kind of problem is; divide your PCR components into 3 sets. One set will be exclusively used for keeping only positive control. Keep that set separately. Use the 2nd set to screen the samples and use the 3rd set to keep Blank and Negative control. Also note that when you are in doubt that the micro-pipette other than the tip portion has touched the wall of the vial; before using it for another component please wipe it once with 70% alcohol and use it after drying.

Is it necessary to keep a blank even when we keep a negative control in PCR?

The answer is yes. Let me explain why. Sometimes we assume a gene to be transgenic and may be it is true. But a similar kind of cisgene may be there which is phylogenetically related to it. For example: Suppose we have a DREB gene from a crop which is more responsive to drought than the given crop in which we need to impart resistance. After introducing the trans DREB gene suppose we went for PCR with a DREB gene specific marker then there are chances you get a same sized band in the negative also. Sometimes when both the related genes are of different sizes then it is also possible that in a transgenic plant you get 2 bands; one for the transgene and the other for the cisgene and in negative you may get only one band corresponding to the cisgene. This is why we keep the DNA of any non-transgenic plant of the same variety or line as negative control for PCR. But the purpose of keeping a Blank (All the PCR components except template) is quite different. A blank can show positive only when there is template contamination from a positive sample or from the positive control in anyone of the PCR components or more than one PCR components. Thus keeping a blank in PCR always helps us to avoid false positives and also helps us to find out whether the components are safe or contaminated.

L:Ladder (100 bp), P: Positive Control (Plasmid) and B: Blank. The figure shows the PCR confirmation of Nos promotor gene (Whether present or not in the plasmid ). The empty B (Blank) Lane assures us that there is no contamination in any of the PCR components used and the result we are getting in the positives are true positives. Amplicon size is nearly 240 bp. Agarose gel electrophoresis done in 1.2% Agarose gel.

Negative 260/280 nanodrop reading for Nucleic Acid means what?

All of us know when we take a reading which can have minute deviations from the original value due to background noise; we do a normal thing, i.e. (S-B) , where S is the sample reading and B is the reading from the blank. Theoretically B should be 0, but as a matter of fact B shows minute absorbance due to presence of some substances those can also absorb at 260 and 280nm. Thus to get the actual amount of substance present in a given sample we subtract the Blank reading value to get the accurate sample reading. Also note that as per expectation the absorbance from the blank is always a minute value never greater than the sample value. A Nanodrop is no different. It also follows the same principle. But for a negative reading to come up, S-B has to be -Ve, which implies the B value has to be greater than S. This can happen only when the 260/280 ratio of the Blank aliquot, which I now refer as the absorbance ratio is more than that of the sample. Usually this happens if the aliquot used as Blank (control) is contaminated with nucleic acids heavily or with some other components having a higher absorbance at 260nm or if the pedestal is not properly cleaned before loading the Blank.

What is the Importance of 260/230 ratio in a Nanodrop reading?

Well, most of us are familiar about the 260/280 ratio.The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA and RNA. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. If the ratio is appreciably lower in either case, it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm. 

But you might have also observed the machine also takes a 260/230 ratio reading but might not have bothered about it because most us only depend on the 260/280 ratio for quality check of nucleic acids(DNA or RNA). This ratio is used as a secondary measure of nucleic acid purity. The 260/230 values for “pure” nucleic acid are often higher than the respective 260/280 values. Expected 260/230 values are commonly in the range of 2.0-2.2. If the ratio is appreciably lower than expected, it may indicate the presence of contaminants which absorb at 230 nm. 

Contaminants like EDTA, carbohydrates and phenol all have absorbance near 230 nm. 

The TRIzol reagent is a phenolic solution which absorbs in the UV both at 230 nm and ~270 nm. 

Guanidine HCL used for DNA isolations will absorb at ~230 nm while guanidine isothiocyanate, used for RNA isolations will absorb at ~260 nm.

Why shouldn't we turn on white light in the laminar airflow chamber immediately after UV treatment?

By fact UV sterillzation is based on the principle of mutation caused by UV C occurring in the DNA of several micro-organisms like bacteria,fungi and protozoa due to thymidine dimer formation(covalent bond formation in between two adjacent thymines in the same strand of the dsDNA which hinders biological processes like cellular DNA replication and transcription)between 245 to 300nm wavelength of UV. This mutation is reverted back by an enzyme called photolyase in the cell which starts working at 480nm(visible region of the spectrum). The phenomenon is called photo-reactivation. If the mutation is fixed then the cells may again start to replicate normally and cause contamination. Thus, it is a general procedure to switch off UV and leave the LFC in dark for at least 15 minutes before turning the white lights on.

Why 70% ethanol is normally used as a sterillant instead of absolute ethanol?

First of all 100% ethanol is highly volatile and tends to evaporate before entering into the tissue significantly. Secondly when absolute ethanol enters the cell first it encounters the cell wall and immediately starts coagulating proteins present in the cell wall before actually considerably entering into the cell and therefore the protective coagulated layer stops further inflow of more ethanol into the cell. In this case the cell is protected and stays dormant and again functions normally when favourable conditions reappear. But if we use 70% ethanol it evaporates much slower than absolute ethanol undoubtedly and it also coagulates proteins and dissolves lipid but at a very slower rate; enough for it to completely get into the cell and then starts coagulating proteins and dissolving lipids and thus the cell dies.why 70% ethanol is standardized for surface sterilization? It may be because perhaps the dilution is enough for entering into the surface layers of the plant tissue where the pathogens may get into and does not go too deep into the plant tissue so that the plant cells beneath the surface tissues are not considerably harmed.

Can the same PCR program have different completion time in different thermo-cyclers?

Yes !!! the same PCR program may have slightly different completion time for the same reaction (having the same number of cycles) in two different thermo-cyclers due to difference in the ramp rate of the machines. For example if I keep two sets of PCR with the same program; one in a Bio-Rad machine and the other one in an Eppendorf machine then one may have a completion time of 1hr 48mins and the other may have completion time of 2hrs 5mins.