PM Sayk and see if he/she worked out the problem.
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There have been 197 items by jerryshelly1 (Search limited from 05-April 19)
It may affect your DNA concentration, but not the residual protein. If they are immune compromised (i.e. low WBC's) you would expect a lower than average DNA concentration, but the overall consistency of the blood would be similar.
As far as the chemotherapy, I do not have enough knowledge on that to make a guess. Are you only seeing this phenomenon in you cancer and chemo patients? Do your healthy controls show this?
I checked the wizard protocol and it is very straight forward. Do you see any clumps in the whole blood before you begin your extraction? Is this a recent problem that was absent in the past?
It was a silly question, but I am just making sure the blood was homogenous before you began your prep. Blood will separate into its respective components fairly quickly and I have seen people take the plasma or the RBC's while leaving the buffy coat almost undisturbed.
What kit are you using? Make sure all your solutions are homogenous. If you continue to have problems, you can contact the manufacturer and see if there have been additional problems with your particular lot number. If anything, they may give you a new kit. I'm not certain though.
It may be worth it to contact another company and see if you can try one of there kits for free. Since you regularly isolate blood, it will be beneficial for them. I have had good success with EZ Bioresearch's MW blood isolation kit (competitively priced).
It makes me worried that two people have commented on the inefficiency of this kit.
Are you using fresh blood and/or are you properly resuspending it before beginning the isolation? If it is older blood, it may contain fibrin which can induce clotting and will make it more difficult to adequately remove all protein.
The only thing I can suggest is taking some "junk" blood and running a couple of different conditions side by side.
Increase centrifugation time on a sample, precipitate protein at 4C for 10min?, increase amount of precipitation solution?
If you are extracting blood, it looks like your protein precipitation buffer is no longer viable. The red color is due to the protein hemoglobin being present in the solution. I would double check your protocol and make sure that you are allowing for proper precipitation of your protein and also check to see if your precipitation buffer is coming out of solution.
Are you centrifuging your samples adequately each time? Is it possible you are overloading the kit with blood each time?
As far as the brown pellet, it looks like you still have cellular debris in your sample.
Sorry to hear that. It is extremely unfortunate that you are in this position. How can this individual maintain her funding if she is an overall terrible mentor? It seems that the administration would look poorly on her overall negligence.
We use those and they work fine. Just practice better aseptic techniques. If it is common lab reagent, make sure everyone is informed on the proper handling of the chemicals. Sounds like someone was careless pouring the solution and introduced a contaminant.
Has this protocol worked before? Homogenization wouldn't be enough to rupture the cells for DNA extraction. Adding sodium acetate will do nothing for DNA precipitation if the DNA is still contained within the membrane. I would add a lysis buffer to your cells and nix the vigorous homogenization.
Edit - Hobglobin beat me to it. Listen to him.
Is this a kit based extraction or a crude phenol-chloroform extraction?
When DNA concentrations are low, you can precipitate your DNA out with 10% NaOAc/EtOH for 30min to O/N at -80C. You also add glycogen if your ethanol solution if your DNA is being difficult.
Edit: Oh the typos...
Silly question, but you are trying to amplify each exon independently correct... Running your FWD/REV primer pairs through NEB and IDT gives a two Tm's that are relatively close. Make sure that your primers and all of your reagents are diluted properly before running and give gradient PCR a try.
It sounds like the annealing temperatures of your primers are way to far off. Does the sequence of ABCD1 make it impossible to design primers that have similiar annealing temps? Are you able to post your primer sequences?
Yes, your control sounds good. Your CIP treated vector with ligase should not religate unless your phosphatase is not working properly; especially with two non-compatible cohesive ends.
How close together are your two restriction sites? 3, 3, 12 bp?
Did you mean you have had success with colony PCR using these primers in the past?
You have a very interesting problem...
Same here. We autoclave regular tap water and supplement the water with SDS. Distilled and Milli-Q water will rust your incubator.
What is the purpose of adding SDS? As an anti-bacterial?
We have seen it reduce the growth of bacteria. It is especially helpful if someone accidently splashes media into the water tray.
There was a paper published a number of years ago showing colony PCR's susceptibility for a high number of false-positives. The authors suggested that the false positives may be the result of the ligated vector + insert's inability to enter the bacterial cell. The correct ligation product would therefore be in your mixture of cells and may "stick" to the outside of your bacterial cell via a LPS. Who knows if this is the correct case, but I know I have had very little success with correctly identifying constructs via colony PCR. I usually avoid this procedure.
In your controls, do you mean that your vector only did not have ligase? It sounded like you just had ligation of your originally cut vector; however, it doesn't make any sense why your vector only would have fewer colonies...