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.