I extracted total DNA of Hedera helix plant's leaves via CTAB method then i used to amplify the matK & rbcL genes at different PCR protocols (Temperature and time) the reaction mix used is Master mix of (DreamTaq Green PCR Master Mix (2X)= 12.5 ul, PCR water 8.5 ul, For Primer= 1.0 ul Rev Primer= 1.0 ul, template DNA= 2 ul)= total= 25 ul reaction.
Primer Used were as follows:
F= 5’-CGATCTATTCATTCAATATTTC-3’
R= 5’-TCTAGCACACGAAAGTCGAAGT-3’
F= 5’-CGTACAGTACTTTTGTGTTTACGAG-3’
R= 5’-ACCCAGTCCATCTGGAAATCTTGGTTC-3’
F= 5’-ATGTCACCACAAACAGAAAC-3’
R= 5’-TCCTTTTAGTAAAAGATTGGGCCGAG-3’
PCR Opt: No of Cycles= 35
95 ℃ = 10 min,
94 ℃ = 30 sec,
55 ℃ = 1 min
72 ℃ = 1 min
72 ℃= 10 min
Storage temp:= 4 ℃
But did not obtained any results (Bands). Can anybody guide me where is the problem with my PCR?
I have also used psbA-trnH and ITS genes for amplification with different PCR temp: but did not obtained any results there as well? Is ther any problem with my Template DNA or something else?
what is the od260/280 of your dna.
When all primer sets fail to amplify then either your dna is low quality or your pcr kit is not working. Borrow a primer set from a friend that works and pcr with his dna and his and your primers and also your dna with his and your primers. How much dna are you amplifying?
often plant dna has pcr inhibitors in it so sometimes amplifying less dna or a re purification of your dna works but most important at this stage is a 260/280 ratio so that you know if your dna is of high quality and good enough for pcr
Greetings,
Besides what Paul says, you could Also try to run your DNA extract on gel electrophoresis, agarose 1.5%, and check if there is any signal of your DNA being lysed. Also, you could try running PCR "universal primers" to amplify the 18s gene, if no band comes up, it's almost sure that your DNA extract is of no Good quality.
Hii,
Firstly, you have to check your quality and quantity of DNA on Nonodrop/agarose gel if it is ok then, Use PCR components instead of master mix (varies Mg ion 1.5-4mM concentration, and use gradient pcr). you will get amplification after the optimization of annealing temp and Mg ion conc
It might be due to either from dna quality or Primer. You may try different primer optimized for the species you are working on.
What is the final concentration of the primers you are using??
Bundle of thanks to Paul Rutland , Adan Valenzuela Castillo , Subhash Kumar and Ferid Abdulhafiz Kemal for your answers bu today I determined the OD 260/280 and I found it totally out of the range.The Values are as below:
Sample 1= 1.02
sample 2= 2.37
sample 3= 1.46
sample 4= 2.57
sample 5 is a bit astonishing one having value= 7.3...
all values are outlier.
Now can anybody suggest me the best DNA extraction method from Hedera helix plant's dry leaves?
CTAB does itself absorb at around 260 so you could try just reprecipitating the dna in salt ethanol and washing twice with 70% ethanol after precipitation and all of the CTAB should wash away leaving a cleaner product
If you suspect there's something in the DNA that's inhibiting the PCR you could try diluting it (e.g. 1/10 and 1/100); we've had success with plant PCRs in our lab doing this. If you have a positive control that's working well (doesn't have to be in this species, any any old PCR will do) you could also try an inhibition test by adding some problematic DNA to the good DNA to see if the PCR stops working. Best of luck.
Paul Rutland and Tania King thanks for your worthy guidance and suggestions.
Possibly PCR inhibitors, the easiest way that worked with us is to bind the DNA to a Qiagen spin column, wash DNA, and elute DNA with ddH2O.
Check your dna concentrations and quality by nanodrop if possible.
check your MM with working dna and primers, because it was happened before with me where I used MM from invetrogen , it was the reason. I didn’t thinkin about MM because it’s from good source.
good luck
Hatem Zayed do you mean DNA binding with the spin column after it is extracted and re-suspended? I f yes please provide me the protocol of DNA binding after completion of DNA extraction, purification and re suspension and secondly tell me please that will any other spin column not work instead of Qiagen beacause its very expensive.?
Thanks for the response.
You may need to check the DNA purity and then add a clean up step by ethanol and heat up. All the best.
We have not tried another spin column, but you can give it a try. The way we did it was to take the already extracted DNA, incubate on a spin column for 1-2 min, just right on the center of the column. Then you spin for 6K rpm for 5 min, then wash 2-3 times with 70% ethanol, leave to dry for 1-2 min, then elute with ddH2O. I hope this works for you, good luck!
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