PROMO is a virtual laboratory identification of putative transcription factor binding sites (TFBS) in DNA

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Plasmid MAXI prep for inVitro translation

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Plasmid MAXI prep

  • Grow E. coli in 100 ml LB + antibiotic (e.g. 100ug/ml ampicillin final).
  • Spin 5000 RPM  or more for 10min and just keep pellet.
  • Add 5 ml of solution I ( glucose 0.450 g , 1.25 ml of Tris-pH8 1M,  1 ml of EDTA-pH8 0.5 M, up to 50 ml water) and vortex to have no clumps.  (50 mM glucose, 25 mM Tris-pH8, 10 mM EDTA-pH8).
  • Add 5 ml of fresh solution II (1 ml of NaOH 10N, 2.5 ml of SDS 20%, up to 50 ml water) and invert gently several times  (never vortex and not more than 5 min). (0.2 M NaOH, 1% SDS).
  • Add 5 ml of ice cold solution III (14.7 g KOAc, 5.75 ml glacial acetic acid, up to 50 ml water) and invert gently several times to mix and put on ice for 10 min.
  • Spin 5000 RPM or more for  15 min. For Vial, 12000 RPM or more.
  • Put a piece of cheesecloth (gauze) at the end of a barrel of a 20 ml syringe and pure the supernatant inside it and install its plunger and push it and collect the pass-through in a tube.
  • Add 0.6 volumes ISO-propanol, invert to mix well and leave 10 min at RT.
  • Spin 4000 RPM or more for 15 min.
  • Re-suspend pellet in 700 ul TE-pH8 and transfer it to a 1.5 vial and add 700 ul of ice-cold 5M LiCl and mix well by inverting and keep on ice for 10 min.
  • Spin 10000 RPM 10 min and keep supernatant.
  • Add 0.6 volumes ISO-propanol to supernatant, invert to mix well and leave 10 min at RT.
  • Spin 10000 10 min and keep pellet.
  • Add 250 ul of TE-pH8 and add 5 ul RNase cocktail and keep at 37 for 30 min.
  • Add 240 ul of Proteinase K buffer and 10 ul/ml of Proteinase K and keep at 37 min.
  • Add 300 ul of phenol-pH8 and vortex horizontally for 15s and keep for 5 min at RT. Then add 100 ul chloroform and vortex horizontally for 15s and keep for 5 min at RT.. Spin 10000 RPM for 10 min and keep supernatant.
  • Add the same volume of ISO-propanol, vortex and keep 10 min RT, then spin 10000 RPM 10 min.
  • Add 1 ml of 70 % ethanol, vortex 5-10s and spin 8000 RPM for 1 min.
  • Dissolve the pellet in 1 ml of sterile water and add 0.5 ml of solution (40% PEG 8000/30 Mm MgCl2). Let at RT >10 min and then spin max speed for 20 min at RT.
  • Re-suspend in 0.5 ml 70% Ethanol and spin again for 5 min max speed. Drain, invert and repeat.
  • Add 200 ul DNase free water or TE-pH8 directly on pellet and after dissolving, transfer it to new vial.

The 10 Worst Things That Can Happen When You Don’t Get Enough Vitamin D

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Do you live in North America? There’s a pretty good chance you’re low on vitamin D. About 42% of U.S. adults are deficient in the nutrient, according to the journal Nutrition Research. And that’s not good. A vitamin D deficiency is associated with a slew of health risks. Plus, if you’re diagnosed with a serious condition such as breast or prostate cancer, your chances of survival may be lower than someone who has normal levels.

No one’s exactly sure why low levels of D are tied with poorer health outcomes. However, experts suggest that the Institute of Medicine’s current recommended daily intake—600 IU for men and women—is simply too low. “Optimal blood levels of vitamin D to reduce your risk of disease is 28 to 42 ng/mL,” says Joel Fuhrman, MD, author of Super Immunity. “Many people need about 2,000 IU of vitamin D3 per day to achieve that level.” He suggests getting your vitamin D levels checked by your doctor in order to determine how much D you need to raise your levels within the optimal range.

Here’s the lowdown on how a vitamin D deficiency can seriously compromise your well-being and take years off your life:

1. You’re more likely to be depressed.

People with low levels of vitamin D in their blood were more than twice as likely to be diagnosed with depression than those with higher levels, according to a study in the British Journal of Psychiatry that included more than 31,000 participants. The hippocampus and other areas of the brain involved in regulating your mood contain vitamin D receptors, so low levels may affect the ability of these regions to function normally, researchers suggest.MORE: 6 Natural Depression Remedies Worth Trying

2. You’re less likely to survive cancer.
Cancer patients who have higher levels of vitamin D when they’re diagnosed tend to live longer and remain in remission longer than patients who are deficient, according to a study published in the Journal of Clinical Endocrinology & Metabolism. Researchers found that every 10-point increase in vitamin D levels was associated with a 4% increase in survival among people with cancer. The strongest link between vitamin D and survival rates were found in patients with lymphoma, colorectal cancer, and breast cancer. In fact, breast cancer patients with healthy vitamin D levels are twice as likely to survive the disease as patients with lower levels, reports the journal Anticancer Research.

3. You’re more likely to develop aggressive prostate cancer.
The risk of aggressive prostate cancer was 4 to 5 times greater in men with low vitamin D levels, according to a study in the journal Clinical Cancer Research. The reason isn’t yet understood, though researchers say that screening for vitamin D deficiency and treating it may become an essential part of cancer care.

4. You have an increased risk of dementia and Alzheimer’s disease.

Adults who were moderately deficient in vitamin D had a 53% increased risk of developing dementia, and those who were severely deficient had a 125% increased risk of the disease compared to individuals with healthy levels, reports a study in the journal Neurology. A deficiency in the nutrient was also associated with up to a 122% increased risk of Alzheimer’s disease. More research is needed to better understand the connection, but researchers suggest that you face a double whammy as you age: Not only are you more at risk of developing cognitive problems, your skin becomes less efficient at converting sunlight into vitamin D, putting you at an increased risk of deficiency.5. You may be more likely to have psoriatic arthritis.
About 30% of patients who have psoriasis also have a condition called psoriatic arthritis, in which the immune system attacks the joints causing pain and inflammation. And a recent study found that up to 62% of people with psoriatic arthritis have insufficient levels of vitamin D, reports the journal Arthritis Care & Research. Previous research shows that low levels of D may make inflammatory conditions such as psoriatic arthritis worse, possibly by increasing white blood cell levels.

6. You may be at risk for more severe heart disease.
People with vitamin D deficiency had a 32% increased risk of coronary artery disease compared to those with normal levels. They were also 20% more likely to have a severe form of the disease, affecting multiple vessels, according to research presented at last year’s American College of Cardiology’s Annual Scientific Session. Vitamin D may improve immune function and control inflammation throughout the body, which can help reduce the risk of heart trouble, researchers say. (Check out 7 more weird ways you’re increasing your risk of heart disease.)

7. You could get pneumonia.

The risk of developing pneumonia is more than 2.5 times greater in people with the lowest vitamin D levels in their blood, researchers at the University of Eastern Finland found. Previous research suggests that vitamin D deficiency weakens the immune system, which increases your risk of contracting illnesses such as respiratory infections.

8. You’re more at risk of being developing schizophrenia.
People with vitamin D deficiency were more than twice as likely to be diagnosed with schizophrenia compared to people who had sufficient blood levels of the vitamin, reports the Journal of Clinical Endocrinology & Metabolism. More research is needed to understand the role vitamin D plays in psychiatric health.

MORE: What It’s Like To Grow Up Terrified You’ll Inherit Your Mother’s Schizophrenia

9. It may speed up multiple sclerosis.
Previous research has found that low vitamin D levels are associated with an increased risk of neuromuscular disorders such as multiple sclerosis, Parkinson’s disease, and others. Now, a new study suggests that if you have multiple sclerosis, low levels of D could speed the severity and progression of the disease, JAMA Neurology reports. Researchers found that early-stage multiple sclerosis patients with adequate levels of vitamin D had a 57% lower rate of new brain lesions and a 57% lower relapse rate than those with lower vitamin D levels. Identifying and treating vitamin D deficiency should become part of the care newly diagnosed patients receive and may actually boost the effectiveness of certain therapies such as interferon beta-1b, the researchers say.

10. You’re more likely to die prematurely.
People with low blood levels of vitamin D are more likely to pass away sooner than those with normal levels, reports an analysis of 32 studies published in The American Journal of Public Health. People with vitamin D levels less than 30 ng/mL had the highest risk of premature death due to all causes than those with levels greater than 30 ng/mL. More isn’t always better: Researchers found no additional benefit for people with levels above 50 ng/mL.

MORE: 17 Surprising Ways To Get More Vitamin D