Cloning guide

 

Vector preparation:

 

Digest 4-6g of vector with appropriate enzymes (see restriction digest protocol)

If double digest not possible purify reaction with PCR-Purification Kit after first digest, then do second digest.

 

Example:      12l plasmid = 5g

                        4l 10xNEBuffer 2

                        4l 10xBSA

                        1l NheI 10U/l

                        0.5l XhoI 20U/l

                        18.5l H2O

 

 

Incubate 1.5 – 2h at 37C.  For cloning excessive reaction time can lead to exonuclease activity.

 

CIAP treatment after digest: can be done in NEB restriction enzyme buffer without adding new buffer.

add 2l of CIAP 1U/l: (stock CIAP 20U/l, dilute with CIAP dilution buffer)                       

for sticky ends incubate 10min at 37C

for blunt ends incubate 5min at 50C

 

 

If restriction digest cuts out fragment smaller than 100bp vector can be purified with either PCR-Purification Kit or with Gel Wizard Purification Kit (useful if elution needs to be done in less than 50l)

If fragment is bigger, separate on gel, cut out appropriate band and purify with Promega Gel Wizard

 

Insert preparation:

 

If PCR amplified (see protocol) and only one single band visible on gel, purify with PCR-Purification Kit

If PCR not very clean and produced several bands separate on gel, cut out appropriate band and use Gel Wizard

 

Digest insert with appropriate enzymes (see above)

 

PCR product: clean and concentrate with gel wizard columns, elution volume 15-50l (no gel purification)

If insert cut out from another plasmid separate on gel, cut out band and gel purify with Gel Wizard

 

Check 2l of purified vector and insert for concentration by running on gel

Estimate amounts and make sure to have one single band each, at appropriate size.

Estimation of amount on gel with kb-ladder: load 0.5g of DNA ladder, 3kb band=125ng

Ladder should be prepared as follows:

Kb ladder: stock 0.5g/l (-20C):            60l stock

                                                                        50l 6XDNA sample buffer

                                                                        190l H2O

final concentration 0.1g/l, store at 4C

load 5l = 0.5g, if concentration of ladder different adjust volume accordingly

 

 

Ligation:

 

Use 3:1 molar ratio of insert to vector taking into account the molecular mass of both

 

(size vector / size insert)/3 = factor F

amount of vector / F = amount of insert

 

Rapid DNA Ligation Kit (read manual before using)

 

For a 20l reaction dont exceed 200ng of total DNA

 

Vector                         x ng

Insert                          x ng / F

DNA dilution Buffer  2l

H2O                             to 10l, mix

T4-Ligase Buffer      10l, mix

T4-Ligase                 1l

 

Control: vector without insert to check for re-ligation rate of vector

Incubate 5-15min at room temperature 

 

If vector/insert/DNA dilution Buffer mix exceeds 10l increase volume to 20l adjusting the volumes accordingly (4l DNA Dilution Buffer+vector+insert +H2O to 20l), add 20l T4-Ligase Buffer +1l T4-Ligase and incubate 30min at room temperature (see manual)

 

Use 2-5l for transformation of 50l competent cells (DH5 alpha subcloning grade)

 

Anne Schohl, 2008