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.
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There have been 197 items by jerryshelly1 (Search limited from 05-April 19)
Can you restate your question? If you are looking for general SNP information, it can be found on UCSC Genome Browser. When you search for a SNP, it will usually give you the allele frequency of that SNP in humans and sometimes other organisms. If you are trying to look at the genomic signatures, then you would probably want the raw sequencing data with the electropherograms. I think it would be best if you were more clear with your question.
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.
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?
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.
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.
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?
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.
Email the company, but yes they should work. Silica columns are not really proprietary and every company uses the same one. The columns may differ in the grade of plastic and the design, but the principle will be the same.
You could nanodrop your PCR sample or use a DNA specific fluorometer for an exact reading; however, using HindIII for densitometery is an alright method.
Increasing the reaction time could potentially help you. You may just run the reaction until the enzymes are dead, but it shouldn't hurt you overall.
Sometimes colleagues prepare the wrong concentration of antibiotics. Double check and make sure you are adding the appropriate concentration.
Can you share the gene you are trying to TOPO?
Edit - One more question... Can you cut out your gene from your other plasmid using a blunt RE? Phusion polymerase is amazing, but maybe something funky is going on.
The ratio refers to the amount of vector to the amount of insert, i.e. 1:1, 1:3, etc...
You have used this kit with the provided control and are sure that it works?
I have used sticky end based TOPO kits and have had varying success depending on the gene. Double check and make sure you are correctly calculating the concentration/ratio of your vector and insert. If varying the ratio of the vector:insert doesn't work, I would consider increasing the TOPO reaction time.
You could feed your insert though a secondary structure prediction program and see if it is forming a weird structure that is impeding proper ligation... but this is probably has nothing to do with your problem.
How many colonies did you test?
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?
You can't run them together. I just open two windows and compare them. If you are looking to find a common Tm, try NEB's Tm calculator.
You could always consult Emsembl or Genome Browser to see relative GC content across your gene of interest.
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.
Solubility of a drug is dependent on the medium it is in. Drugs show different solubilities in water, alcohol, etc.. Drugs are also suspended in different mediums depending upon the drugs mode of entry. If it is via skin, you will need medium that is fat soluble. If it is oral, water soluble can suffice and so on...
When you are finished with your transfer, stain your gel with commassie to see if any residual protein remains on the gel. Either your proteins are not transferring or your proteins are blowing through your membrane.
Maybe there is allele variability for that particular amplicon region. You could run the samples on a gel and see what the size difference is. If you see two distinct bands, you probably have allelic variation, if you see a smear adjust your cycling conditions. Are these previously established qPCR primers? Taqman?