I would suggest use as little as the colony for PCR (using a point toothpick). How much did you take from a colony to do the PCR? Alia Mike

Considering your PCR protocol is correct & standardized (annealing temperature & time & elongation time), absence of product suggest problem with the template. I completely agree with @Sayanta Bera in terms of cell lysis before proceeding to pcr. You may isolate the plasmid DNA from the cells & then use it as template for pcr.

Dear Talia

I'm agree with Sayanta, it seems that the PCR do not work! I do not think is a problem related to the Safeview stain because the marker is clear detectable.

Which PCR protocol did you used and how did you prepared the samples?

How long is the PCR insert that you would like to amplify with the screening?

If you are interested on my blog ProteoCool

https://proteocool.blogspot.com/

you can find at PAGE1 - ProteoCool n°4 where you may find the details of the protocol that i'm routinelly using for PCR colony screening.

Ciao

Manuele

Alia Mike As your ladder is clearly visible, there is no problem with the stain.

How sure you are about the colonies that you got are positive? I mean to say, have you checked that only host cells do not grow on selective media plate? If you have taken all the controls and sure about the transform ants then problem is with the PCR.

During PCR, do not use colony directly in PCR tube. Instead, in 50 uL of milli-Q water dispense the cells from colony using strile toothpick. Heat this at 95 C for 5 min, allow it to cool and centrifuge at max speed for 5 min. From supernatant use 5-7 uL as template. Provided, you have standardise protocol for colony PCR, this would work. Just be sure that you are keeping elongation time for 2 min per kb of amplicon.

Hi Alia Mike indeed it seems that the PCR was not successful. Assuming that you had made the necessary adjustment regarding the time and temperature for each step of the PCR, there are some other things that might affect the efficiency of PCR:

- the presence of amplification inhibitors in the genomic DNA. This can be solved by reducing the volume of template DNA in the sample, or by diluting the DNA prior to adding it to the PCR master mix.

- presence of impurities. This can be solved by including an ethanol precipitation step and washing steps before the actual amplification

- sometimes adding PCR additives such as DMSO or stabilizing agents such as BSA, might help.

Hope this helps!