VDS of the FRI
Summer 11
Ligation with simultaneous Restriction Enzyme Digest and Transformation
ProtocolCloningLigate_REdigest_Transform_pUC19VDS_Summer11.doc
Purpose: to join Insert and Accepting Vector together
These modifications to the original protocol will make the process longer but hopefully improve
probability of success. Instead of carrying out the first part of the procedure in one day, the prepared
PCR product and prepared Vector can be saved - 20oC overnight.
Carry out PCR2 Squared to amplify many copies of your gene (do multiple samples if
desired)
Gel Purify PCR product (2-3 hrs):
To minimize contamination of your DNA, use clean reagents
Make a 0.9% Agarose gel (0.9 grams of agarose in 100 ml of TAE)
Run 100 bp ladder
Skip a lane
(so that you don’t mix ladder with your sample)
Run your PCR Squared product
Place on top of saran wrap on imaging box
Image gel (quickly – to reduce UV light damage to DNA)
Excise band with razor blade
Carry out Gel Extraction kit – elute in 30 ul of buffer without EDTA (eg. Tris is ok)
Phosphorylate PCR product
X
ul
PCR product (use all)
5
ul
10X T4 PNK Buffer
5
ul
10 mM ATP (1 mM final conc.)
X
ul
DDW to 49 ul
1
ul
10 Units of T4 Polynucleotide Kinase (T4 PNK)
50
ul
Total Volume
Gently pipette up and down to mix
Spin short
Wrap top in Parafilm to seal and place in floater in water bath
Incubate at 37oC for 30 minutes
Carry out PCR cleanup column - elute in 30 ul of buffer without EDTA (eg. Tris is ok)
Nanodrop to determine concentration (Can Store in - 20oC overnight)
To save time, the following can be done simultaneously with the above procedures
Digestion of Accepting Vector (1.5 hrs):
Make 2 new 1.7 ml centrifuge tubes (control tube, experimental tube)
1,500 ng
plasmid
2.5
ul
10X Restriction Enzyme Buffer (determine which you need for your RE)
X
ul
100X BSA (determine if recommended for your RE)
X
ul
DDW to 24 ul
1
ul
10 Units of Restriction Enzyme (select one: -- SmaI or HincII)
25
ul
Total Volume
Gently pipette up and down to mix
Spin short
Wrap top in Parafilm to seal and place in floater in water bath
Incubate at 37oC for HincII or 25oC for SmaI for 1-2 hours
Incubate at 65oC for 20 minutes to kill enzyme
(Can Store in - 20oC overnight)
1
VDS of the FRI
Summer 11
Ligation with simultaneous Restriction Enzyme Digest and Transformation
ProtocolCloningLigate_REdigest_Transform_pUC19VDS_Summer11.doc
OPTION: CIP or AP
Dephosphorylation of vector (< 1 hr):
 Calf Intestinal Phosphatase (CIP)
Use same tube as the Restriction Digest
1
ul
10 Units of CIP
o
Incubate at 50 C for 30-1 hr
Carry out PCR cleanup column directly after
dephosphorylation – elute in 30 ul
Dephosphorylation of vector (< 1 hr):
 Antarctic Phosphatase (AP)
Use same tube as the Restriction Digest
3
ul
10X AP Buffer for Zn
1
ul
10 Units of AP
Incubate at 37oC for 30-1 hr
Incubate at 65oC for 5 minutes to kill enzyme
Don’t have to do PCR cleanup
Nanodrop to determine concentration
(Can Store in - 20oC overnight)
Prepare 2 Xgal/IPTG plates with Ampicillin for Blue/White Screening
(one for experimental, one for control)
Ligation (1.5-2.5 hrs): (Ligation will now be a standard ligation)
Ligation should be done right before Transformation
Prepare 2 tubes (one for experimental, one for control)
Calculate amounts: Need (1:3) ratio of accepting vector to insert (e.g. 50 ng: 150ng)
Expt Ctrl
X
X
X
X
X
1.5
1.5
ul
Add in accepting vector (to both tubes)
ul
Add in PCR product (to experimental tube only)
ul
10X Ligase buffer (keep ligase buffer on top of ice – to stop ATP breakdown)
ul
T4 DNA Ligase to both tubes (experimental and control)
30
ul
Total Volume for each
Let tubes sit at room temp (20-25OC) for 2 hours
Spin down brief
65oC heat block for 10 min to stop reaction by killing enzyme
Spin down brief
Proceed immediately to transformation
Transformation with highly competent cells (2 hrs):
(control + experimental)
15 ul of each ligation reaction into 25 ul competent cells
NEB DH5α High Efficiency Competent Cells (only remove 1 tube of 50ul of competent
cells because you can split it in half – half for control, half for experimental)
 Store leftover ligation rxn at -20oC in case you need to redo
 if too many colonies – can lower ligation amount next time
Mix by tapping tubes gently
Incubate on ice for 30 minutes
Heat shock for exactly 30 secs in 42oC water bath
2
VDS of the FRI
Summer 11
Ligation with simultaneous Restriction Enzyme Digest and Transformation
ProtocolCloningLigate_REdigest_Transform_pUC19VDS_Summer11.doc
Place back on ice
Add 175 ul of prewarmed SOC (using sterile technique)
Shake for 1 hour in 37oC shaker
Spread all of transformation mixtures to plates with colirollers or metal loop
Incubate plates upside down overnight at 37oC
Next Day
- in the morning ~ 9 – 10 am
Storage and growth of colonies
The master plate is basically an archive of your colonies so that you can go back to it and re-pick
the correct ones. Master plate does not need to be Blue/White (i.e. Xgal,IPTG)
Pick ~8 colonies from original plate and simultaneously
1. make Master Plate with numbering grid (with appropriate antibiotic)
2. ~8 tubes with 5 ml of LB with appropriate antibiotic to grow up the colonies
When picking the colony – dab the location on the master plate first, then go to the
correspondingly numbered tube and drop the toothpick/pipette tip into the tube of LB.
#1
#2
#3
#4
#5
#6
#7
#8
1. Grow up tubes during day (8 hrs) in numbered tubes
a. Use 37oC shaking incubator.
2. Store original plate in 4oC wrapped in parafilm to seal it
3. Place Master Plate in 37oC stationary incubator to grow o/n
6:00 – 7:00 PM: At end of day, spin down and save samples
Next day, do Miniprep Kit on samples to extract DNA for testing
– elute with water at pH 8.0 or 5 mM Tris-HCl, pH 8.0 (avoid TE because of the EDTA can
interfere with downstream reactions – e.g. DNA Sequencing)
Nanodrop to determine concentration of DNA
Pick two good ones and send to DNA sequencing as final check and to determine if mutations are
present - Use M13 Forward (-40) and M13 Reverse (-48) primers
Alternatively, or in addition – can carry out RE digest if you have enough DNA to quickly verify
positive clones.
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