need to make sure the process work, I want to be sure that the amplicon I obtained was good enough for clone library construction. Hence I wish to know much much quantity of PCR product (ng/microlitre) is optimum for clone library construction? I got faint bands in PCR (12.5 ul total volume, 2ul is used for gel check), confused if this is ok for clone library construction.
the quantity of DNA to be used depends on the Vector to insert ration. larger the insert size more the DNA concentration per ul you require. I suggest you to calculate the molar concentration of your DNA insert to optimize the vector to insert ration as desired.
1) Measure your PURIFIED PCR product [ng/uL] - nanodrop, spectrophotometer, etc.
2) Calculate vector AND insert into pmoles in 1ul (molarity of 1ug dsDNA:
[pmoles]=3360,4x[bp]^(-1,0123) (r^2=0,9999)
3) Use proper molar ratio of vector and fragment(insert) DNA:
a) Sticky ends: when DNA and insert DNA are ~similar in length: a molar ratio of 1:3 (vector vs. insert DNA); when not similar in length: ratios 1:1 or 1:2 should be used
b) Blunt ends: ratio 1:5.
What kind of library are you trying to make? If you are actually trying to make a library (as opposed to generating a single plasmid), then you will need enough primary clones after transformation to ensure that your library is complex enough.
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