General Caution:Make sure your bench has a clean diaper for ALL PCR steps. Use filter tips for everything—if reagents get contaminated with an old PCR product or clone, you could spend weeks following up on false positives, or tracking down the source of the background.1. Primer Design- (Outside Users provide their own primers.)The choice of gene-specific primers for your screen is done in three steps: Step 1: Use primer design programs to choose gene-specific primers that will amplify sequences in the first ~1 kb of your gene. Order 3-to-5 primers that point upstream toward the promoter region, and 3 or 4 primers that map in, or close to the promoter region and point downstream. Step 2: Test each possible pair of opposing primers in PCR reactions, to identify primer pairs that successfully amplify the target gene. Purify and sequence one of these fragments, and use it to create a digoxigenin probe for the screen. Step 3: Run each primer that passed Step 2 through the gamut of controls described below, in which their compatibility with the Mu primer and other related issues are explored Each of these steps is explained in detail below. A. The Mu Primer:The Mu Primer pair below binds to the terminal inverted repeats of all members of the Mu family. We use a mix of two primers. The working concentration for each one is 25µM, which gives a total concentration of 50µM Mu primers.:
B. Gene-Specific Primers:WE RECOMMEND THAT YOU DESIGN AT LEAST 4 PRIMERS IN EACH DIRECTION AT THE OUTSET, BECAUSE PRIMERS OFTEN FAIL THE CONTROLS DESCRIBED BELOW. Choose primers that will allow you to "scan" as much of your target gene as possible. PCR from each primer should scan at least 1 kb; insertions as far as 3 kb from a primer have been detected. Mu elements frequently insert near transcription start sites. Therefore, you may be more successful in identifying an insertion in your gene if you screen PML with a GS primer that points towards the promoter (such as primer B in the diagram). In fact, the majority of the Mu insertions found through forward genetic screens map either within the promoter or less than 800 bp downstream. Therefore, we highly recommend that you start by screening with a primer that points upstream, and that maps ~800-1000 bp downstream of the transcription start site (see primer B in diagram)
Unfortunately, some wild-type genes appear to have Mu-TIR-like sequences
in their promoter regions and therefore give amplification products
when a Mu primer is paired with an upstream-pointing primer. If this
is a problem in your gene, it will be revealed by the control reactions
described below. In this case, you could use a primer that maps near
the transcription start and points downstream (like primer A in the
diagram). Simply cut and paste the available gene/cDNA sequence into the window, we have set the defaults appropriately for PML. Checking Primers: You need to check that the primers chosen by Primer 3 will not bind at spurious locations or form primer dimers. To do this, we use the program Amplify (http://engels.genetics.wisc.edu/amplify/ ). Paste your sequence into the sequence window and your primers into the primer list window. Then choose all primers to be used in the PCR reaction and run your PCR. The program will give you an output window that shows whether the primers are likely to mis-prime or to form dimers. If primers are predicted to show spurious binding, try to eliminate this by shifting their positions slightly. If you can’t get rid of it, throw the primer away. Do not worry about primers that are predicted to form dimers unless the pair will be used in the same reaction. We are happy to discuss primer design with you should you have trouble meeting the above criteria (request@pml.uoregon.edu). 2. Testing Primers in Control PCR reactionsOnce you have designed primers you need to test them. This is done in two steps. Step 1: Set up PCR reactions containing each primer pair that will give you a product between 100 and 4000bp. The results will tell you which primer pairs work together.
*Add Primers separately (0.5µl each gene-specific. One pair per reaction). Thermocycler Profile:
Run 20 µl of PCR product on a 1.2% agarose gel and check that the primer pair gave you the expected product size based on sequence. Step 2: Choose ~three of the successful primer pairs for the subsequent set of control reactions (part b below). Use primer sets that will amplify the first 1 kb of the gene, if possible. It is useful to test several “3’ primers” (those pointing upstream) in conjunction with a 5’ primer in the promoter region, just in case one control is not clean. The ideal screen uses an upstream pointing primer that binds ~900 bp downstream of the transcription start site. b). Prior to screening the PML library your paired gene-specific primers need to be tested for (i) specificity and (ii) yield. In addition, your gene-specific primers paired with the Mu primer need to be checked to be sure there is NO background amplification of your target gene when DNA from "wild-type " Mu-active maize lines is used as template. Below, the general PCR protocol is listed, followed by a description of each control reaction. ControlsWe recommend using Ex-Taq (Takara) polymerase for consistency. This is supplied with its own buffer and dNTP mix by the manufacturer. PCR Conditions
*For screening the PML library, one of the GS Primers is replaced with the Mu primer. Thermocycler Profile:
**note that the annealing temperature of this reaction can be increased if spurious amplification is observed.
Controls to test the gene-specific primers:Set up the following five controls in parallel, and probe a Southern blot of the products with a target gene probe of confirmed sequence. (do NOT assume that a PCR product generated by paired GS primers on genomic DNA corresponds to the correct gene- the product MUST be sequenced before using it to generate a probe). (see Probe preparation). Summary of the controls: 1) Use GS primer pairs to amplify a portion of your target gene. 2) Use the same gene-specific primer pairs in a reaction without any added genomic DNA. Southern blotting, using the probe described above should give no signal. This will reveal whether you have plasmid/PCR product contamination in your stocks and if problematic primer dimers are created with your primer pairs. 3) Test each GS primer with the Mu primer and 50 ng of
wild-type DNA from a Mu-active plant. This should give NO signal when a Southern blot of the products is probed
with your gene probe. 4) Test each GS primer individually with 50 ng of wild-type
DNA from a Mu-active plant. This should give NO signal when a Southern blot of the products is probed
with your gene probe. If signal is detected, the primer is mispriming at a second
site within your gene. This could interfere with your screen by creating false
positives. 5) Test that the Mu primer does not inhibit the ability of the GS primers to
prime. After you have identified a set of GS primers that pass all controls, if possible,
start your screen with a primer pointing 5' (upstream) and that maps ~500-1000
bp downstream of the transcription start site.
Required DataIn order to evaluate your controls, and to compare to our results with your primers, we need to see these controls. Ideally we would like an image of the gel with the above 7 lanes on it, and an image of the blot generated by transfering that gel, and probing with your biotintylated gene-specific probe. We're quite flexable on file format, but would prefer that the image(s) have the lanes, the primer pair, and the probe clearly labled. Note Mu insertion preferences: All the successful PML screens was have done to date have yielded insertions within ~800bp of the start codon, but this data is biased, because we have generally been LOOKING in the first 1000bp or so. The insertions obtained through forward genetics do not have this bias.
see blotting and hybridization protocols and Probe preparation for further details. |
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