The overall goal of the following experiment is to generate transgenes using worm Genomic DNA carried in a SMID library clone. The use of genomic DNA preserves all of the native control elements such as enhancers in three prime UTR regions. This is achieved by moving the FO smid into the SW 1 0 6 E coli strain to utilize the inducible lambda red recombination machinery.
As a second step, the GAL K gene is inserted into the desired location in the FO smid, which marks the site where the GFP or tap tag will be placed. Next, the Gade gene is replaced by GFP or a tap TAC via homologous recombination, followed by negative selection using deoxy galactose in order to create the modified transgene. Results are obtained that show these transgenes can be created quickly and successfully based on verification of T insertion by colony PCR.
Hi, my name is Love Ship. I work in the Fisher Lab at University of Pittsburgh. Today I'm demonstrating the technique from making C elegant transgene.
Why recombining with GK as the selectable marker. The main advantage of this technique over existing methods of creating Cogan Transgenes is that in this technique we use the genomic DNA of the gene of interest and thus are able to cover all the critical elements of gene regulation such as control elements in three prime UTR region, alternative spice transcripts, alternative promoters, and distal answers which might be missed out in conventional standard cloning based approaches. So let's get started.
Order the FO smid clone for the gene of interest or GOI from gene service using Worm Base as a guide. When selecting clones, we choose ones that have the GOI in the center of the sequence. Clones that exclude neighboring genes might be preferable, but could be hard to find culture.
The SMID clone for the GOI in LB containing 12.5 micrograms per milliliter chloramphenicol, the smid are sent in the EPI 300 strain and can be grown at 37 degrees Celsius. Next, grow a 1.5 milliliter overnight culture of the SMID at 37 degrees Celsius and mini prep the smid DNA. Using the Epicenter SMID prep kit, we follow the alternate protocol described in the instructions, which involves adding the Ribo shorter mix At an earlier step, determine the FOSS DNA concentration.
Using a spectrophotometer field is usually very low but adequate. For successful electroporation. Prepare electro competent SW 1 0 6 cells by growing a five milliliter overnight culture in LB media at 32 degrees Celsius in a 14 milliliter snap cap tube.
The following morning. Inoculate one milliliter of the culture into 100 milliliters of lb in a two liter flask. Grow SW 1 0 6 bacteria to an OD 600 of 0.6 to 0.8.
Do not heat shock. Pellet the cells by centrifugation at 5, 000 Gs for five minutes. Re suspend the pellet by gentle vortexing in one milliliter of ice cold 10%glycerol.
Then add 50 milliliters of ice cold, 10%glycerol. Repeat this wash step once. Finally pellet the SW 1 0 6 by centrifugation again and DEC can't All but about 500 microliters of each supernatant Resus.
Suspend the pellets by gentle vortexing freeze 100 microliter Ali Watts in liquid nitrogen or on dry ice and store at minus 80 degrees Celsius until ready for transformation. Transform the fosson DNA into electro competent SW 1 0 6 cells by electro parading the bacteria with about 50 nanograms of fos DNA in 0.1 centimeter gap cuvettes using an EOR 25 10 electro perter at 1, 350 volts. Recover the bacteria in one milliliter of LB for one hour at 32 degrees Celsius plate Eloqua on LB plates with chloramphenicol and incubate at 32 degrees Celsius overnight.
Verify the presence of the gene of interest using colony PCR by growing a five milliliter overnight culture in LB with 12.5 micrograms per milliliter chloramphenicol at 32 degrees Celsius. Next at 0.5 microliters of the culture to a standard PCR reaction with the flanking oligos and increase the initial 95 degrees Celsius incubation to five minutes to slice the bacteria prior to the reaction. Finally, prepare a glycerol stock for long-term storage to insert the gal K gene into the FO smid carrying your gene of interest.
First, prepare mops minimal media plates containing 0.2%galactose. See the written portion of this protocol for the recipe. Next PCR amplify the P mod four gal, k, g or P mod four gal K GT cassette.
We use fusion or go tac gel. Purify the resulting PCR product. Quantify the yield on a gel or by using a NanoDrop spectrophotometer.
Store the PCR product at four degrees Celsius until ready to use inoculate five milliliters of and chloramphenicol with SW 1 0 6 cells containing the Foss DNA grow overnight at 32 degrees Celsius. Add one milliliter of overnight culture to 100 milliliters of lb and chloramphenicol. In a two liter flask grow to an OD of 0.6 to 0.8.
This usually takes three to four hours while the culture is growing. Set a shaking water bath to 42 degrees Celsius to warm up with a sterile 250 milliliter flask in the holder. Use of a shaking water bath for the next step is critical for obtaining high efficiency.
Next transfer 50 milliliters of the SW 1 0 6 culture to the 250 milliliter flask and heat shock at 42 degrees Celsius and 100 RPM for exactly 20 minutes in the shaking water bath. Leave the remaining bacteria at 32 degrees Celsius to use as the un induced control. Cool the induced and un induced bacteria on ice for 10 minutes.
Transfer the samples to two sterile centrifuge tubes and pellet at about 5, 000 Gs for five minutes. Pour off all of the supernatant and resuspend the pellet in one milliliter of ice cold 10%glycerol by gentle vortexing. Add another 49 milliliters of ice cold, 10%glycerol.
Repeat this wash then pellet once more. Remove the supernatant by inverting the tubes and resuspend the pellet in the small amount of remaining liquid Eloqua into 100 microliter samples. Freeze on dry ice and store at minus 80 degrees Celsius the following day.
Electro parade, the induced and un induced SW 1 0 6 cells with 150 nanograms of the prepared PCR product using 0.1 centimeter gap cuvettes in an einor 25 ton electro ratter. Set at 1, 350 volts. Recover the bacteria in one milliliter of LB in a 14 milliliter Falcon tube.
Incubate at 32 degrees Celsius for four and a half hours after the incubation pellet the bacteria at 13, 200 RPM for 15 seconds and resuspend in M nine medium. Please refer to the written portion of this protocol for the recipe. Repeat this wash Step two times.
To remove any rich medium, pellet the cells once more than resuspend in one milliliter of M nine before plating serial dilution on mops. Minimal medium incubate three to five days at 32 degrees Celsius in an incubator. Be patient as the true positives grow slowly streak a colony on Macon agar to purify the colony and verify that it is GLK positive.
The colonies should turn bright pink. Pick a single colony and inoculate a five milliliter lb plus chloramphenicol overnight culture incubate at 32 degrees Celsius. Confirm insertion of the gal caging at the proper location via PCR using the flanking oligos.
Refer to table one in the written portion of this protocol. For the oligo sequences, add 0.5 microliters of the culture to a standard PCR reaction and increase the initial 95 degrees Celsius incubation to five minutes to ly the bacteria. The PCR product should be upshift in size due to the presence of the GAL K gene.
Finally, prepare glycerol stock for storage. Prepare mops minimal media plates containing 0.2%deoxy galactose or DOG and 0.2%glycerol. See the written portion of this protocol for the recipe.PCR.
Amplify the tag fragments from PMO 4G FP PBS 1761 or PBS 1479 using the same oligos used in the first round or using shorter GFP or tap specific oligos. If you are making multiple constructs, it is particularly useful to use the shorter oligos as the same PCR product can be used for all of the constructs Gel. Purify the PCR product and measure the concentration on a gel or with a spectrophotometer generate induced and un induced competent W 1 0 6 cells carrying the SMID with the Gale K gene as described earlier, ate the induced and unin induced SW 1 0 6 cells with about 100 nanograms of PCR product using 0.1 centimeter gap Q vets in an EINOR 25 10 ator set at 1, 350 volts.
Recover in one milliliter of LV in a 14 milliliter snap cap tube and incubate in a 32 degree Celsius shaker for four and a half hours. Wash and dilute the cells in M nine as shown earlier. Plate the bacteria on mops, minimal medium containing 0.2%DOG and 0.2%Glycerol incubate the plates at 32 degrees Celsius for three days to confirm the presence of the insertion.
Use four colonies to inoculate five milliliter overnight cultures in LB with 12.5 micrograms per milliliter. Chloramphenicol perform colony PCR as described earlier. We use both the shorter GFP tap specific oligos and the flanking oligos to demonstrate the right insert and right site.
GFP is about 800 base pairs. TAP is about 550 base pairs and gal KT is 1.4. Kilobases finally prepare a glycerol stock for long-term storage.
The modification of FO smed via recombining gives robust results and success rates of greater than 90%in the negative selection step are routinely observed. In addition, this protocol takes about two weeks to complete, which makes the preparation of transgenes fairly rapid, shown our 48 colonies identified on DOG plates and used for colony PCR using the flag GFP Oligos, which amplified the GFP insert in this experiment. 47 outta 48 colonies are correct and one colony is not correctly modified shown are 48 colonies identified on DOG plates and used for colony PCR using the flag GFP Oligos, which amplified the GFP insert in this experiment.
46 out of 48 colonies are correct and two colonies are not correctly modified. The most important point to keep in mind while performing this experiment is the SW 1 0 6 bacteria has to be grown at 32 Celsius and not at higher temperatures because growth at higher temperature will select mutants with mutations impairing the Lambda red machinery. So best of luck with your experiment.
