Insert Check

Plasmid mini-prep is a technique which is rarely done in my lab. Plasmid mini-prep is done only when the circular plasmid is necessary for some experiments. Instead, it is enough to get sequence data from colony direct PCR products. Recent advancement of sequencing technology made it easier to read templates not very clean such as enzymatically purified PCR product.

Insert Check PCR Premix (for 1mL)


dNTPs (2mM each; final 0.2mM)        100uL
M13FW.HT primer (100pmol/uL)        5-10uL
   (CGC CAG GGT TTT CCC AGT CAC GAC)
M13RV.HT primer (100pmol/uL)        5-10uL
   (AGC GGA TAA CAA TTT CAC ACA GGA AAC)
10 x KOD-dash buffer                 100uL
KOD-dash (Toyobo, Japan)               5uL
   (increase up to 20uL according to the insert length expected)
milli-Q water                        780uL

Make up the reaction premix and store in a freezer. Stable for about one year. Amount of enzyme added to the premix is much smaller than a standard reaction condition, and the buffer and dNTP mix will exhaust quickly. The manufacturer of KOD-dash (Toyobo) supplies additional buffers and dNTPs free of charge upon request.

Picking Colonies

Dispense 10-15uL of reaction premix onto 96-well PCR plate, etc. A smaller reaction volume (10-15uL) is enough. Scaled-down reaction with lower concentration of enzyme makes at most 20 times more ecnomy than the standard condition. Pick up single colonies and soak the needle into premix solution for a couple of seconds. Use of 0.2mm plutinum needle is convenient, because it cools down rapidly after burning from colony to colony and enables quick picking many colonies.



Cut the plutinum needle just to reach the bottom of the PCR well, then it is unnecessary to watch the bottom of well when you soak the needle in the reaction mixture.

PCR Cycle

300 sec at 95oC
  v
20 sec at 98oC
5 sec at 60oC
120 sec at 72oC
(30 cycles)
  v
60-300 sec at 72oC

Extention time is 1 min per kb of expected product length in the standard condition, but for economy reactions it is better to employ 2 min per kb to obtain good amounts of product.
Samples sequenced with BigDye kit according to this protocol and enzymatic purification showed bad peak separation. Frequently it is unreadable beyond only 300 bases. BigDye kit seems vulnerable to KOD-dash carryover, and good sequencing performance recovers upon PK treatment to kill the enzyme. After 30 min incubation of PCR product with ExoSapIt at 37C, add 0.1uL of PK (dilute for convenience) and incubate at 37C for 30min more, and then heat inactivate for 15 min at 80C.
On the other hand, ET kit is tolerable to KOD-dash and gives good separation over 500 bases. For better performance, kill the enzyme as above. I recommend to use ET kit to get longer read (>700) of inserts, because BigDye kit shows still lower peak separation beyond about 600bp even upon PK treatment for KOD-dash carryover.

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