I am trying to clone a 3.8Kb gene into a 5.7Kb vector ( pFBDM ), here is my protocols:
10ul of vector was cut with 1uL of XmaI and KpnI ( NEB ) in Cutsmart buffer for 3 hrs at 37degC ( 50uL total reaction volume )
Restrictions enzymes are fresh stock purchased about 1 month ago.
Gel purify the cut plasmid. ( UV was avoided by cutting the gel at the corresponding site )
purified PCR product was cut with 1uL of same enzymes in same buffer for 3 hrs then remove the enzymes with Qiagen kit
50ng of cut vector was used in ligation reaction
vector/insert ratio: 1/2
ligation O/N at 16 degC ( NEB T4 ligase )
*****Note: I included a negative control
cut vector+ ligase but NO insert added
plate on selective plate and culture O/N at 37 degC
negative control plate: 3 colonies yielded
experimental plate: around 50 colonies
I picked up 8 colonies and do overnight culture. I cut the purified plasmid with two enzymes but no insert found!
Need your opinions.