I am going to design two oligonucleotides (the size about 20 base) which can be annealed together forming two sticky ends then will be ligated to the digested vector with the corresponding enzyme. Does anyone have the protocol steps?
The protocoll could be the following.
Add equal amount of both primers (you can use 100 ng of each), heat them at 80 ºC for 5 min, remove them and let them coll down to room temperature for 10 -15 min.
You can run the sample in a 2-2,5% Agarose gel to make sure it worked ok. Et-Bromide only stains dsDNA so it is usefull to check the anneling. You can also load the individual primers to check that they give no signal. Load bewteen 10-50 ngr, do not exceed this amount to have nicer bands.
Remember that the anneled product will NOT have phosphates so you will need to add the phosphate with a T4 kinase prior to ligation (just follow the protocoll recomended by the enzyme). If the sticky ends correspond to the same restriction enzyme you should de-phosphoriate the vector prior to ligation.
Then you can ligate your insert. You will probably have to dilute your anneled product a lot in order to have vector:insert ratios of 1:1 or 1:3 or 1:10.
Finally consider that for such small inserts there is a high chance of getting tandem repeats of your insert so be carefull with your screening.
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