I am currently investigating the CRISPR 9 case to target my gene of interest for knockout. The vector being used is lentiCRISPR v2puro. I digested and dephosphorylated 5ug of the lentiviral CRISPR plasmid with BsmBI,Subsequently, I ran a gel electrophoresis as shown in the attached image.
Following this, I excised the larger band and stored it at -20°C overnight. The next step involved purifying the gel using a Promega kit. During the purification process, I added 500ul of membrane binding solution, briefly vortexed the mixture, and then incubated it on a heating block at 88°C for approximately 25 minutes until the gel slice completely dissolved.
The DNA concentration for 5ug was measured at 30.144 ng/μl, with a 260/280 ratio of 1.70 and a 260/230 ratio of 1.26.
Next, I performed phosphorylation and annealing of each pair of oligos, which were then diluted to a 1:200 ratio. The two target sequences are as follows:
I have heard that it is recommended to add "g" to target sequences even if they already start with "g", as omitting it could result in a lack of colonies after transformation. is that correct ?
The third step involved ligating the sequences using Quick Ligase. In the ligation reaction, a 1:3 ratio was used, so 2.2 ul of vector and 6.8 ul of insert DNA were added, along with 10ul of ligation reaction buffer and 1ul of Quick Ligase, making the total reaction volume 20ul. The mixture was then incubated at room temperature for 1 hour before adding 50 ul of DH5a cells. Heat shock was performed by incubating on ice for 30 minutes, followed by a 30-second incubation in a 42°C water bath and then back on ice for 2 minutes. Subsequently, 250 ul of prewarmed LB media was added, and the mixture was incubated on a shaker at 37°C for 1 hour. The resulting solution was then spread onto LB agar plates containing Ampicillin (100 μg/mL) and incubated in an incubator at 37°C overnight. Colonies were obtained for the positive control using pUC19, but not for the sample.
During this this procedure If anyone has any insights into why colonies were not obtained your input would be greatly appreciated.
"I have heard that it is recommended to add "g" to target sequences even if they already start with "g", as omitting it could result in a lack of colonies after transformation. is that correct ?" That is incorrect. You only add G to a target spacer because transcription of the human U6 promoter is more efficient if there is a G in that spot. Omitting it would only lower abundance of guide RNA transcribed in human cells (lowering the targeting efficiency). The U6 promoter does not work in E. coli.
I have two guesses as to why this didn't work:
1. If you phosphorylated the primers with NEB's T4 Polynucleotide Kinase and used the PNK Buffer that comes with the enzyme, the reaction will not work. They leave the required ATP out of the buffer so people can add radiolabeled ATP, mostly to make radioactive DNA probes. For routine phosphorylation, you have to use the regular 10X T4 Ligase Buffer, which includes ATP.
2. If your gel was illuminated with UV light, you probably overexposed it and damaged the vector too much to be replicated. 88°C for 25 minutes in a denaturing buffer also sounds excessive for melting the gel and might have impacted the DNA integrity somewhat.
Alexandra Johnson thank you so much for your response, I will give try for your suggestion for the phosphorylated.
In this part ( During the purification process, I added 500ul of membrane binding solution, briefly vortexed the mixture, and then incubated it on a heating block at 88°C for approximately 25 minutes until the gel slice completely dissolved), Sorry for the typo in my previous statement regarding the temperature, the temperature was 68 C for 25 minute, not 88C for 25 minutes, Is it still sounds excessive for melting the gel?
Thanks again Dr. Alexandra Johnson
68° for that length of time should be fine. When you're cutting the band out of the gel, is the gel illuminated with UV light, or are you using blue light?
Actually, the gel was exposed to UV light for a few minutes to visualize the band and cut the band.
If possible, I would get a stain like SYBR safe where you can illuminate the bands with blue light which won't damage the DNA and you can take your time shaving off extra gel, in my experience this usually results in purer DNA as well.
If there's no blue light transilluminator around, then next time I'd work as quickly as possible on the lowest power and longest wavelength setting the UV transilluminator has to limit damage.
Alexandra Johnson Thanks again Dr. May, I would like to know the recommended volume of SYBR safe stain to add when using 1.5% agarose gel made by mixing 0.45 g of agarose powder with 35 ml of 1x TEA buffer.
Thank you for your help.
I think it's usually sold as a 10000X stock in DMSO, so 3.5 µL. If you have trouble seeing the band then double it to 7 µL.
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