Preparing a Sample for Submission
For most applications the user supplies RNA or DNA, while the unit performs the specific processing of the sample as required, hybridization of the labeled sample to the array of choice (which is purchased directly by the user), scanning of the array, quantification of the image and basic analysis. Advanced analysis is not supplied by the unit, but can be obtained from the Bioinformatics unit (**link!)
Input quality: Sample purity and quality is of top importance. The rule of 'Garbage-In-Garbage-Out" applies to all assays and arrays. It is worth putting effort in preparing good input samples to significantly increase the chance of obtaining good results. High quality input material means DNA or RNA which is
- Intact (not degraded). This can be tested by gel (RNA and DNA);
- 260/280 ratio of 1.9-2.1 for RNA, or 1.7-1.9 for DNA
- 260/230 ratio not lower than 1.5. This is important - consult us if you cannot reach those figures.
- RNA/DNA extractioni should be carried out using any kit/procedure that works well for your cells/tissues. There is no specific recommendation as long as the RNA/DNA meets the criteria. If you are using organic solutions for extractions (phenols), be sure your 260/230 ratios are high, as phenol carry-over will lower the ratio and disrupt any downstream reactions. NOTE: The quality of the input material is the responsibility of the user! Samples which will fail to be successfully labeled due to low-quality input material will still be charged!
- Unless otherwise specified, samples should be submitted in nuclease-free water. Most extraction kits supply such water.
- Submit your samples in 1.5 ml tubes for all applications. Make sure tubes are well labeled and that labels are easily understood.
- RNA samples should be submitted on ice or dry ice, but not in liquid nitrogen.
- When submitting samples, please print out the relevant submission form, fill it out and bring it with your samples. Samples will not be accepted without a form. * INSERT SAMPLE SUBMISSION FORMS!
- Required amounts and concentrations by application: When submitting your samples, make sure to follow the specifications listed below. In case of need to de viate from these figures, please consult us first.
Affy Gene/Exon ST Input: Total RNA Total amount: 100-250 ng Concentration: 100 ng/ul Volume: Minimum 5 ul 260/280 ratio: 1.8-2.1 260/230 ratio: preferably higher than 2, not less than 1.5 Affy 3' (using IVT-Express kit) Input: Total RNA Total amount: 500 ng Concentration: 200 ng/ul Volume: at least 4 ul 260/280 ratio: 1.8-2.1 260/230 ratio: preferably higher than 2, not less than 1.5 Affy miR Input: Total RNA Total amount: 1 ug Concentration: minimum 150 ng/ul Volume: maximum 8 ul 260/280 ratio: 1.8-2.1 260/230 ratio: preferably higher than 2, not less than 1.5 Affy SNP6 Input: Genomic DNA Total amount: 1 ug Concentration: 100 ng/ul exactly Volume: >10 ul 260/280 ratio: 1.7-1.9 260/230 ratio: preferably higher than 1.5 Agilent expression Input: Total RNA Total amount: 200 ng Concentration: 100 ng/ul Volume: 5 ul 260/280 ratio: 1.8-2.1 260/230 ratio: preferably higher than 2, not less than 1.5 Agilent CGH Input: DNA Total amount: 0.5-1 ug Concentration: minimum 25 ng/ul Volume: maximum 20 ul 260/280 ratio: 1.6-1.8 260/230 ratio: preferably higher than 1.5 Agilent miR Input: Total RNA (Do not use the size fractionation or small RNA enrichment protocol) Total amount: 100 ng Concentration: 50 ng/ul Volume: 5 ul 260/280 ratio: 1.8-2.1 260/230 ratio: preferably higher than 2, not less than 1.5 TapeStation RNA Input: Total RNA Concentration: 2-500 ng/ul Volume: minimum 3 ul TapeStation DNA Input: DNA Concentration: 75-1000 pg/ul Volume: minimum 3 ul Fluidigm Real-Time qPCR Input: cDNA and primer pairs Concentration: 50ng/µl cDNA and 20µM primer pairs Volume: at least 6µl (in 96-plate)
Blue Pippin - Reagents and Cassettes are provided by the users (available at Zotal Ltd)!
Sample should be in a 30µl TE Buffer:
Maximum Load: 10g sheared genomic DNA
2 g restriction/PCR band capture
Minimum Load: low single nanograms, sheared genomic DNA
50 ng restriction/PCR band capture
Optical Sensitivity approx. 10-15ng single bands, restriction
(Midori-Green cassettes): fragments, PCR products, or synthetic DNA fragments
Input Sample Characteristics
When running the BluePippin, characteristics of input DNA can affect separation resolution and efficiency of product recovery. The following general guidelines should be followed:
- Ionic strength: The ionic strength of the sample should be lower than the ionic strength of the buffer (80mM monovalent ions). High salt concentrations can result in slower than expected DNA mobility.
- Protein in the sample: DNA-binding proteins such as ligases or polymerases can affect the mobility of fragments during separation. Proteins can also reduce DNA recovery from the elution module by increasing the binding of DNA to the ultrafiltration membrane at the back of the elution module. For best results, samples should be de-proteinized prior to loading whenever possible.
- Input DNA size distribution: A knowledge of the input size distribution is obviously important to program accurate size selection settings. BluePippin cassettes are calibrated using the Agilent Bioanalyzer to evaluate input and product sizes, and so, for best results, input size distributions should be evaluated using the Bioanalyzer. For low concentration samples, the Agilent HS chip is very useful .