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Genetics/In vitro transcription


Dear Dr. Robichaud,
I don't know if you can help me with this question (if not, please forward it to one of your colleague experts). I am a PhD student and have some trouble with in vitro transcription. I did several T7 transcription and they always yield in the same concentration (even when you try different amounts of DNA template and even if you use water as template). After IVT, I perfom a DNase treatment and a purification using magnetic beads that capture the RNA. But still, my negative control sample (water) gives a signal by nanodrop which resembles RNA.
Do you perhaps know what is going on here?
Thanks in advance for your help.
Kind regards

Hi Marleis,

I did one rotation in an RNA lab back in the day. Working with RNA is a pain because it is so easily contaminated. I have a few cleaning recommendations, many of which you've probably already tried. Also try reaching out to the older students in your department/university who work with RNA.

In short, RNA in your water control usually means contamination. Clean, clean clean.

1) Do you only use RNA pipettors for RNA work? If not, do you wipe down and sterilize the pipettors *every time*, even as far as sucking up solution inside the barrel of the pipettor? (Inside the pipettor is a famous place for random, annoying contamination.)
2) Do you wash your hands well before starting? Wear gloves? Lab coat? Face shield or mask? Wearing all these when handling samples helps.
3) Do you buy RNA free water for this reaction, and use it for only this?
4) Do you wipe down your station BEFORE and AFTER all RNA work? Do you wipe down your equipment BEFORE and AFTER all RNA work?
5) Do other people use your RNA kits? Cite contamination issues, and buy your own fresh one. Guard it jealously. Only use that.
6) Only use sterile (autoclaved) tips. If you rack your own tips, try using only the first half of the box for sterile work and switch to a new box. Or re-autoclave them. Never leave tip boxes open for more than seconds if you can help it.
7) Same rule for solution bottles. Never leave them open for longer than you can help.

Good luck and I hope something in here helps.  


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Trista Robichaud, PhD


No homework questions, especially ones copied and pasted from textbooks. I will answer questions about principles or give hints, but I do not do other's homework. I'm comfortable answering basic biochemistry, chemistry, genetics, and biology questions up to and including an undergraduate level of understanding. This includes molecular biology, protein purification, and genetics. My training/inclination is primarily in structural biology, or how the shapes of things affect their function. Other interests include protein design, protein engineering, enzyme kinetics, and metabolic diseases such as cancer, atherosclerosis, and diabetes. Regrettably, I cannot diagnose any disease. I can tell you how we currently understand the basic science behind a disease state, but I cannot recommend treatment in any way. Please direct such questions to your medical professional.


I hold a PhD in Biomedical Science from the University of Massachusetts Medical School in Worcester. I specialize in Biochemistry, with a focus on protein chemistry. My thesis work involved the structure and functions of the human glucose transporter 1. (hGLUT1) Currently I am a postdoc working in peptide (mini-protein) design and enzymology at the University of Texas Health Science Center in San Antonio, Texas. I am in Bjorn Steffensen's lab, researching inhibitors of gelatinase A, a matrix metalloproteinase. I have also been answering Chemistry/Biochemistry questions on this site since summer 2010.

2001 American Association for the Advancement of Science, 2007 American Chemical Society 2007 Protein Society 2011 UTHSCSA Women’s Faculty Association

Publications Levine KB, Robichaud TK, Hamill S, Sultzman LA, Carruthers A. Properties of the human erythrocyte glucose transport protein are determined by cellular context. Biochemistry 44(15):5606-16, 2005. (PMID 15823019) Robichaud TK, Appleyard AN, Herbert RB, Henderson PJ, Carruthers A “Determinants of ligand binding affinity and cooperativity at the GLUT1 endofacial site” Biochemistry 50(15):3137-48, 2011. (PMID 21384913) Xu X, Mikhailova M, Chen Z, Pal S, Robichaud TK, Lafer EM, Baber S, Steffensen B. “Peptide from the C-terminal domain of tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) inhibits membrane activation of matrix metalloproteinase-2 (MMP-2)” Matrix Biol. 2011 Sep;30(7-8):404-12. (PMID: 21839835) Robichaud TK, Steffensen B, Fields GB. Exosite interactions impact matrix metalloproteinase collagen specificities. J Biol Chem. 2011 Oct 28;286(43):37535-42 (PMID: 21896477) Poster Abstracts: Robichaud TK, Carruthers. A Mutagenesis of the Human type 1 glucose transporter exit site: A functional study. ACS 234th Meeting, Boston MA. Division of Biological Chemistry, 2007 Robichaud TK, Bhowmick M, Tokmina-Roszyk D, Fields GB “Synthesis and Analysis of MT1-MMP Peptide Inhibitors” Biological Chemistry Division of the Protein Society Meeting, San Diego CA 2010 Robichaud TK; Tokmina-Roszyk D; Steffensen B and Fields GB “Exosite Interactions Determine Matrix Metalloproteinase Specificities” Gordon Research Conference on Matrix Metalloproteinase Biology, Bristol RI 2011

INSTITUTION AND LOCATION DEGREE (if applicable) YEAR(s) FIELD OF STUDY Oakland University, Auburn Hills MI BS 1993-1998 Biochemistry University of Massachusetts Medical School, Worcester MA PhD 2001-2008 Biochemistry & Molecular Pharmacology University of Texas Health Science Center, San Antonio TX Postdoc 2009-Present Biochemistry

Awards and Honors
1998 Honors College Graduate, Oakland University 2009 Institutional National Research Service Award, Pathobiology of Occlusive Vascular Disease T32 HL07446 2011 1st Place, Best Postdoctoral Poster, Dental Science Symposium, UTHSCSA, April 2011

Past/Present Clients
Invited Seminars: Robichaud TK, Fields GB. “Synthesis and Analysis of MTI-MMP Triple Helical Peptide Inhibitors” Pathology Research Conference, University of Texas Health Science Center San Antonio Pathology Department (June 18th, 2010) Robichaud TK & Hill, B “How To Give A Great Scientific Talk” Invited Lecture, Pathobiology of Occlusive Vascular Disease Seminars, UTHSCSA (Nov 11th 2010), Cardiology Seminar Series, Texas Research Park (Feb 21st, 2011) Robichaud TK; Tokmina-Roszyk D; Steffensen B and Fields GB “Exosite Interactions Determine Matrix Metalloproteinase Specificities” Gordon-Keenan Research Seminar “Everything You Wanted to Know About Matrix Metalloproteinases But Were Afraid to Ask” Bristol, RI (Aug 6th, 2011)

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