Hello everyone,
I am trying to clone the gene of interest for the mRNA hybridization ( in situ)
after cloning, and miniprepre and restriction digestion, . I don't get the desired product on the gel .
only the vector size on the gel
I followed these steps:
1. PCR run
2. gel cut and extraction by Qiagen kit
3. Ligation : 5ul 2X ligation buffer ( promega), 3ul PCR product,1ul pGEMT easy vector( promega), 1ul ligase, 1hr at room temperature incubation.
4.plated on 100ul DH5-alpha cells
I have plated only on Ampicillin plates and also on X-GAL (150ul) and IPTG (50ul) on each plate . I have colonies on both plates. but they are very small in size . Are they non-specific colonies? size of the blue colonies and white colonies are same ( quite small) .I picked white colonies from ( Amp,X-gal , IPTG plates) and did miniprep then Restriction digestion. I don't see any insert into the plasmid.
I don't understand where the mistake is. Can anyone please guide me .Thanks!
If the blue and white colonies are similar size then it should be ok, a few extra hours in the incubator would let them get bigger.
How big is your insert? Was the plasmid mini prep good so that you say plenty of vector size DNA?
Did you have enough DNA in the mini prep that you would see the inserts well? Those are going to be much fainter than the vector band (especially the 500).
Also did you do a control ligation without insert and what did those plates look like?
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