Cloning, restriction digestion and DNA labeling of large DNA fragments (greater than or equal to 1 kb) in the presence of remelted SeaPlaque GTG agarose gels.
Large DNA fragments (greater than or equal to 1 kb), separated in low melting temperature SeaPlaque GTG agarose gels, can be enzymatically processed directly in the presence of this agarose (in-gel). Time saving protocols are discussed for in-gel processing of large DNA fragments in the presence of remelted SeaPlaque GTG agarose, including cloning into pUC18, nick translation, random priming and restriction digestion. These in-gel molecular biology techniques are as efficient as those using DNA recovered from agarose. The effects of UV irradiation, Mg2+ concentration and agarose concentration on selected in-gel protocols are also discussed.
Daum
H A
HA
FMC Bio-Products, Rockland, ME 04841.
White
H W
HW
Seidell
C M
CM
Johnson
P A
PA
D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D015247:Deoxyribonuclease HindIII / Q000378:metabolism; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D008970:Molecular Weight; D010957:Plasmids / Q000528:radiation effects; D012685:Sepharose; D013696:Temperature; D014466:Ultraviolet Rays
D004247:DNA; D012685:Sepharose; D015247:Deoxyribonuclease HindIII