Epigeneticist

Use the lower phase, which is the phenol/chloroform. The upper phase is water + buffer, holding the pH of the reagent at the correct value. Don't use plastic serological pipets, which will dissolve in chloroform. Polypropylene or glass is fine. You don't need to heat it to RT.
Read further, especially about safety, here:
http://openwetware.org/wiki/Phenol

Phenol extraction and Ethanol Precipitation of DNA
This protocol describes the most commonly used method of purifying and concentrating DNA preparations using phenol extraction and ethanol precipitation; it is appropriate for the purification of DNA from small volumes (<0.4ml) at concentrations ≤1mg/ml.
Materials
-DNA to be purified (≤1 mg/ml) in .1 to .4 ml volume
-25:24:1(v/v/v) phenol/chloroform/isoamyl alcohol (made with buffered phenol: Support Protocol 1)
-3M sodium acetate, ph 5.2
-100% ethanol,ice cold
-70% ethanol, room temperature
-Ultra pure water
1. Add an equal volume of phenol/chloroform/isoamyl alcohol to the DNA solution to be purified in a 1.5-ml microcentrifuge tube.
2. Vortex vigorously 10 sec and microcentrifuge 15 sec at room temperature
3. Carefully remove the top (aqueous) phase containing the DNA using a 200 microliter pipettor and transfer to a new tube. If a white precipitate is present at the aqueous/organic interface, reextract the organic phase and pool the aqueous phases.
4. Add 1/10 volume of 3M sodium acetate, pH 5.2, to the solution of DNA. Mix by vortexing briefly or by flicking the tube several times with a finger
5. Add 2 to 2.5 vol (calculated after salt addition) of ice-cold 100% ethanol. Mix by vortexing and place in crushed dry ice for 5 min or longer.
6. Spin 5 min in a fixed-angle microcentrifuge at high speed and remove the supernatant.
7. Add 1ml of room temperature 70% ethanol. Invert the tube several times and microcentrifuge as in step 6.
8. Remove the supernatant. Allow to air dry for 15 minutes
9. Resuspend DNA pellet in 100 microliters of Ultra pure water
Protocol adapted from:
Ausubel, F.; Brent, R.; Kingston, R.; Moore, D.; Seidman, J.G.; Smith, J.;Struhl, K. Short Protocols in Molecular Biology(1995), 3rd ed., Unit 2.1: page 2-3.

How long are the sequence between the primers when you sequenced them? For any sequence you got from sequencing, can you try to blast them against other species such as human to see if they are contamination? I guess you either have contamination of your PCR system (in this case, you should discard everything and start over), or have non-specific binding to other location(s). Try do a in silicon PCR using your primer sequences to find out. For this, you can lower the criterion for Min Perfect Match.

Even if it degrades, the amount of EtBr needed for staining is miniscule -- much much less than you are adding.

There would possibly be some photo-degradation of the EtBr with time, but this should be fairly minimal unless the time elapsed is substantial (i.e. months).  EtBr will also migrate under an electric field towards the negative electrode (cathode), so technically you will deplete the Etbr in the tank at the anode (+ electrode).  You can just add EtBr to the anode to overcome this problem.  The rate at which it migrates and hence depletes will depend on how long you are running the tanks for and how much charge (voltage) you are putting over them, so there is no "you need to add this amount at X time points" sort of answer.

p53 is normally pretty low expression and very very quickly degraded (30 min half life).  Try UV irradiation (100 mJ in a stratalinker will work, look for p53 within 8 hours) or drug induced for a + control.

At what temperature is the most ideal to store prepared protein samples? -20C? -80C? and for how long?

It definitely depends on the protein, things like actin will last several (10?) but others won't even last 1 cycle.

Either is fine, basically they both work out at the same molarity of reducing molecules despite being different concentrations (DTT has 2 S; B-ME, 1).  DTT tends to go off quicker in storage (stock solution), but smells less (IMO) than B-ME.

In theory you don't need to re-heat the samples after freezing, but many people re-boil them.

Both are fine at 90-95 or 100.  However, the temp you should use depends on the protein you are looking for, some are damaged by high heat.

just reduce the quantity of marker you loading, you can reduce its signal, alternatively if you have extra well just skip a well and start loading the samples...may be your target antibody might not be picking signal in a short time, so your longer incubation and more marker loaded are the possible reason for this.. definitely your actin shd be good, so you wnt get signal dominance frm the marker...

We have had also problems with mouse cells contaminating human cells. In addition, we obtained once a mouse cell line that turned out to be a dog cell line. Since then we regularily check our cell lines for interspecies contaminations. We found a service lab called Multiplexion to do it at a very reasonable price. At the same time they also check for SMRV and Mycoplasma.

I think that if you just use the upper and lower SD of the target and divide by /mean ref copy number, it should be OK.  I don't think the SD of the reference plays a role in this, but I'm no statistician by any means.

Depending on the cell lines you are using just find a mouse-specific or a human-specific gene.  Go to pubmed, find gene and design primers.

You can't do a visual check to see if your cells are contaminated?  Not the best method, but I don't understand why you are doing this.

Just a quick note: there are several versions of both the M13 forward and M13 reverse primers, which differ significantly in sequence. Use care.

The map is OK.

The M13 primers are common for many (if not almost all current vectors) and have a historical identical notation 'M13 forward', 'M13 reverse'.
If those primers are actually in forward or reverse orientation on a plasmid map have no importance at all.
In TOPO cloning the insert is ligated randomly in both orientations (the more it doesn't really matter which primer you call forward and which reverse). So in the one clone you sequenced, insert was in "reverse" orientation regarding to the map.
That's all.