It depend on how big is your DNA (genomic or plasmid) and how dry it is. If your DNA is over dried, it's more difficult to dissolve completely. Bigger DNA will be more difficult to dissolve than smaller DNA. High temp and longer time will help ensure complete dissolving of your precious DNA. If this happen, it will not affect much in your PCR since usually PCR only need very small amount of the template. But keep in mind if you try to measure OD 260 nm of the uncompletely dissolved DNA, you may not get accurate quantification estimate. Make sure there is no visible pellet in your tube. In contrast, if your DNA is not dried enough, the time and temperature for dissolving is not a problem (your DNA will be completely dissolve in no time), but the residual ethanol will have bad effect in your PCR, it can inhibit your PCR reaction.
Hi, Noha, which kit have you used? If they recomenned 90 minutes, you should do this 90 minutes. However, in many case even 30 minutes is enough. You can made spectrum of the DNA after isolation to estimate DNA quality.
I recommend to use TE pH8.0 buffer (if you want to ensure that you must prepare it before DNA extraction), and the time depends directly with the DNA amount isolated. If you washed your DNA ethanol 70% (v/v) solution you have to check that the solution actually was ethanol added prior to use, the absence of ethanol in the DNA washing solution could arise problems with DNA yields.
Drying is to be made sure so that after ethanol washing, there is no ethanol left over in the pellet as it may affect the downstream reactions like PCR etc., If it is dried appropriately, its OK. If its not dried that will effect further. Better is after adding TE or water to dissolve the DNA pellet, check the quality of the DNA in spectrophotometer mostly people use Nanodrop. A 260/280 gives the quality of DNA. If it is in prescribed limits your DNA Is good and you can go ahead
It depend on how big is your DNA (genomic or plasmid) and how dry it is. If your DNA is over dried, it's more difficult to dissolve completely. Bigger DNA will be more difficult to dissolve than smaller DNA. High temp and longer time will help ensure complete dissolving of your precious DNA. If this happen, it will not affect much in your PCR since usually PCR only need very small amount of the template. But keep in mind if you try to measure OD 260 nm of the uncompletely dissolved DNA, you may not get accurate quantification estimate. Make sure there is no visible pellet in your tube. In contrast, if your DNA is not dried enough, the time and temperature for dissolving is not a problem (your DNA will be completely dissolve in no time), but the residual ethanol will have bad effect in your PCR, it can inhibit your PCR reaction.
It is better to follow manufecturer's instructions. An occasional finger tapping will help in quick resuspension of DNA. The best way of checking the complete re-suspension of your DNA is pipetting, if there is a blob seen while pipetting means your DNA is not completely resuspended. in that case you need more incubation time. Icomplete suspension of your DNA , I dont think is going to affect PCR amp;lification. The desired quantity can be used after quantification specterophotometrically.
That should not be a problem. What could potentially be a problem is the solution you used to rehydrate the DNA in. The 90 minutes at 60°C is not so critical.
I think that you have very large amounts of high molecular DNA. I don´t know the kind of sample and kit that you are using. However, my experience say that for DNA re hydratation 60° for 90 min is to much. We use usually 50° for 1 hour.
In my opinion, you can get your DNA but the amount may be lower than in 90min.Most of the factors can effect the yields of your DNA,such as how many nts of your DNA, the amount of DNA in your original material.i have no idea what kit you used,actually it is a long time cost you 90 min getting your DNA . you can check your DNA yields by PCR.if you have aplyfied, checkit by AGE.
PCR as a very sensitive method should not depend too much on this rehydration step, but at the end the quality of the DNA represent a critical issue for very long amplimers.
As much as your kit recommends 90mins rehydration time at 60oC, note that durations of 30-60mins at the same temperature have been successfully used many times depending on certain conditions of the isolated DNA. Whether or not the PCR will be affected also depends on the ultimate purpose of amplification.
There is no doubt that PCR depends on the quality of DNA. For some reactions like AFLP, kits are not recommended. No problem with SSR. Regarding your question, of course the step of Rehydration has indirect effect on the quality of PCR reaction. Most studies recommend to follow the rehydration time (at least 30-60 minutes), but slow rehydration cause the DNA pellet is too dry and in this case you can increase the incubation time and tap the bottom of the tube occasionally to facilitate rehydration. There are some DNA Rehydration Solution that can added for 60 min at 65°C or for 20 minutes at room temperature, or at 4°C overnight.
You could also check the concentration using a nanodrop it provides quick result. Generally speaking, i will advice you stick to the protocol as indicated by the manufacturer, because usually a lot of due diligence goes into it.