Hello
I am having a trouble with the result of agarose gel electrophoresis of the PCR product of Entamoeba gingivalis
DNA
Samples:
Paper point samples from the sulcus(between tooth & gingiva)
Stored in a PBS transition solution
DNA extraction:
1-samples heated to 37˚for 10 mins and mixed well on vortex.
2- 0.3ml of the suspension was washed 3 times with distilled water and spin down between each wash at 14000 rpm for 3 minutes
3- the pellets were re-suspended in 100 microlitre nuclease free water.
Pcr procedure:
1- Primers forward and reverse specific for E.gingivalis were used
2- GoTaq green master mix was used
3- The master mix consisted of the following:
A- 150 µl master mix(GoTaq green)
B- 6 µl forward primer
C- 6 µl forward primer
D- 114 µl nuclease free water
Divided on 12 samples =23 µl
Add 2 µl of each specimen to be the total of the mix 25 µl
4- The reaction cycle was prepared as following:
A-Initial denaturation 95˚ 2minutes
Then 30 cycles of the following
B- 95˚ 30 second
C- 55˚ 30 second
E- 72˚ 35 seconds
Final extension 72˚ for 5 minutes
Question is the following image of the electrophoresis is typica
Thanks in advance
update:
the expected size of the PCR product is
135
in the previous image
today i changed the voltage to 80
and the running time to 40 minutes
and got the following result.
Named IMG_3106
the first 10 lanes are the pcr products
11th is the ladder
20th negative control
update:
20 oct 2024
thanks alot for all the significat paticipation
the agarose gel is prepared by adding 1.5 gram powder to 100ml of TBE (1X) and 5µl of gelred
attached a new picture named IMG_3249
is a photo of the electrophoresis
of a positive control by doing
pcr for RNA 16 s
thank you
the last row is the negative control
It looks like a gel that has solidified but has not completely set. Let the gels cool for longer and lower your running voltage to avoid heating the gel
What size product did you expect? It would also help to label the image to know which are unknowns and which are the controls.
I'd suggest running for a longer time at a lower voltage to get better separation of the ladder (I'm assuming that is what is in the right two lanes).
Good luck!
you may have PCR products in lanes 6, 9 and 10, the rest looks like just the cloud of primers, without any product being amplfied. However, you can't be sure because of the bad quality of the experiment (to short running time, too high voltage, possible air bubbles at the edge of gel, ...)
As noted we need to know expected sizes of PCR product and what samples are found in each lane. What percentage gel?
Thanks all for your significant input and help
i updated the info
and the agarose gel result
Paul Rutland
Joseph Gerard Lorenz
Johann Heider
Katie A S Burnette
the separation is getting better so I would reduce the voltage again and run less sample.......some of the smiling and smearing can be caused by too much salt in the sample.The ladder shows that the gel is good quality. Sample 20 worries me more.It looks like you have contamination with pcr product as there seems to be a band at over 100 bp which is not primer dimer. You need to check its size because if there is contamination then all of your results will be over amplified and of no value as the contamination will dominate the reaction
It seems that the PCR reaction is incomplete, check the annealing temperature by using the gradient and see the difference
Thanks alot all for your significant
input .
updates have been added
Paul Rutland
Suhail Shafi Lone
The ladder and the samples are now running well in the agarose so well done . I would like to see a pcr run with 3 samples which have no added dna ( negative cntrol samples ) because the amplification in sample 20 worries me that you may be generating results which come mainly from only one sample and the end results may be impossible to interpret if you do have pcr contamination
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