Ligation Protocol

2015-01-13

azim58 - Ligation Protocol

Ligation Protocol 081511
Reaction Contents
Total Volume: 20 uL
smaller volumes are actually better (5 or 10 uL reaction); in order to
create such a small reaction it may be necessary to ethanol precipitate
all of the the DNA reactants. The concentration of the DNA can be
estimated by assuming that 90% of the DNA present before the ethanol
precipitation is present.

DNA

Digested Fragment
Digested Plasmid
Shoot for 20-50 ng (for a library may want to use up to 200 ng DNA)

(actually Kathy mentioned I should use 50 ng per 10 uL for a good
ligation) total DNA in a manner such that there is a 3:1 molar ratio of
insert to vector so that there is more insert than vector.
o Spreadsheet for calculating ligation
reaction:"C:\kurt\storage\CIM Research
Folder\DR\2012\10-10-12\Ligation\Ligation Calculator.xlsx"

Note one might want to do an additional reaction with only plasmid

and no fragment to measure the effect of plasmid self ligation.
2 uL 10X T4 DNA Ligase Buffer

last seen -20 #62 2nd shelf top left rack 2nd box back

1 uL T4 DNA Ligase

we use Promega T4 DNA Ligase

0.5 uL 70 mM ATP (not dATP!)

Andrey suggests it is best to keep this ATP in small aliquots and

don't use them more than 2 or 3 times. The ATP improves the ligation
efficiency because the ATP in the buffer degrades after many
freeze/thaw cycles over time.

last seen in 70 mM ATP Stock box in -80 C (#77 2nd shelf from bottom)

(originally Andrey's box)
y uL H20
Quick Ligase Incubation: Incubate at RT for 10 min
Promega Ligase Incubation: Incubate 15 C for 16 hours then hold at 4 C in
a thermocycler
Optional heat inactivation step for some ligases (for example, 80 C 20
min)

edit
Precipitation Protocol After Ligation
Note that whenever I have precipitated DNA, I cannot obtain a good
reading on the nanodrop. I am not sure if this is because of the leftover
glycogen or I may not have let the ethanol dry away completely.

Add water, acetic acid, a carrier molecule, and ethanol

o Start thawing glycogen and ultrapure H20 from kit
- glycogen last seen on Felicia's -20 C shelf) or Andrey's
strategic reserve box -20 C

o Transfer volume to larger tube (1.5 mL)
o Increase volume to 100 uL with water
o Add 1/10 volume (10 uL) 3 M NaAc pH 5.2 (last seen Felicia's
shelf at room temp)
- The sodium acetate acts as a salt to help precipitate the DNA
or RNA

o Add a carrier molecule
- Either
@ Add 2 uL 20 ug/uL glycogen
@ Note that if you need to scale up the reaction for a
larger volume, still just use 2-3 uL of glycogen even though
everything else will be scaled up

- Or
@ Add tRNA to a working concentration of 10-20 ug/mL
# Some people feel that tRNA is a higher quality
carrier than glycogen. It has the advantage of not interfering
with interactions between DNA and proteins like glycogen does,
but has the disadvantage of interfering with any reactions
catalyzed by polynucleotide kinase or terminal transferase.
# tRNA last seen 10-3-11 KW -20 C box

o Add 3 volumes (300 uL) 95-100% ethanol
o Vortex to mix everything up

Cold Precipitation

o Place tube at -80 C for 30 min- couple of hours or -20 C
overnight/over weekend

Centrifuge 14,000 rpm 4 C 15-20 min

o 20817 rcf in Eppendorf centrifuge at room temp next to Debbie's
bench. The current centrifuge in cold room may not spin fast enough
so it may be best to use the one at room temp

Decant supernatent
Briefly spin down again in a small centrifuge. Decant supernatant.
Wash pellet

o Wash with 70% ethanol (with about 100 uL).
- If the pellet is small (like a pellet from about a 20 uL
ligation reaction), you may want to transfer the liquid and pellet
to a smaller PCR tube at this point since the pellet will only be
resuspended in 1 uL.
@ Make sure pellet gets transferred. May need to pipette up
and down to loosen pellet from surface. May want to use 1 mL
pipette tip.
# Note that the 5 mL pipette tips cannot fit into 1.5
mL tubes

- If the pellet is large (like the pellet from about a >50 uL
ligation reaction), you may want to leave the pellet in the same
tube it is in since it is harder to transfer the pellet without
losing some of it, and you may want to finally resuspend in a
larger volume (like 2 uL).

o Gently flick tube to wash pellet
o Centrifuge at 14,000 rpm @ 5 min
- PCR tube can be centrifuged by placing in a 0.6 mL tube
placed in a 1.5 mL tube

o Carefully remove supernatent
o Briefly spin down again in a small centrifuge. Decant supernatant.

Air dry for about 10 min (pellet goes clear)

o May want to air dry in hood to avoid any contamination

Resuspend in 1 uL water (maybe 2 uL for a larger pellet from a >50 uL

reaction)
o May want to use ultra pure H20 from a 1.5 mL tube.
o Note that if the sample was resuspended in a larger volume than 2
uL that the sample concentration can be determined with a nanodrop if
desired. (see determining concentration of dna for further notes)

May want to transform bacteria with the ligation product if that is

the next step for the procedure.
o Electroporation or Chemical Transformation Protocol

===========================================================================
Questions about Protocol
Is it possible to do an in-fusion reaction after an ethanol precipitation?
Yes it is. I have evidence of an in-fusion working after an ethanol
precipitation here: 1-23-13 Sequencing results

A Ligation Spreadsheet
Ligation.xls
"C:\Users\kurtw_000\Documents\kurt\storage\CIM Research Folder\DR\2014\01-23-2014d2247\01-23-2014d2247\Ligation.xls"