Plan for Cloning Mouse scFv DNA 082411

2015-01-13

azim58 - Plan for Cloning Mouse scFv DNA 082411






Plan for Cloning Mouse scFv DNA 082411


Vanessa's spread sheet of work done so far:
ScFv in PCANTAB - digestion, dephosp, ligarton 8-25-2011.xls
Vanessa's spread sheet of digest and dephosphorylation of PCANTAB and
rCOMP TT (kind of general):
Digest - PCANTAB and rCOMP TT with SfiI and NotI with
dephosphorylation.xls

Samples

o see Final scFv DNA 11xx10


Protocol Outline

o Include PCANTAB5E Single Cut, and PCANTAB5E Uncut on gel
o Restriction Digest SfiI and NotI 082411.xls
o Restriction Digest SfiI and NotI 090711.xls
o Cut with SfiI (30 uL volume, Buffer 4, 500 ng vector)
o Cut with NotI (60 uL volume, Buffer 3)
- Note that a test of NotI restriction digest was performed to
make sure that the enzyme and conditions were working.


o Our SAP is from Promega

o Prepare large 0.8% agarose gel (about 350 mL) with 2.4-3 uL of 1%
EtBr
- Andrey commented that if I wanted, I could use about 2/3 of
the gel
- Note that Kathy mentioned using high quality low melt agarose
for this gel extraction, but Andrey says the standard Seakem
agarose we've been using recently works great.

o Place gel in 4 C to solidify fully
o Load samples along with 1 kb ladder
o Run gel at 100 V for about 3-4 hours (maybe even longer (possibly
like 6 hours) for really good separation of cut and uncut vector).
- Andrey thinks it is good to run the gel slowly for a very
long time to make sure that the uncut vector is separated from the
cut vector well.

o Andrey commented that a little bit of ethidium bromide can be
added to the buffer on the positive side of the gel to keep it
running from positive to negative and to prevent a "shadow" from
being visualized later. Just use about 1-2 uL after about 1 hr.
o Take a picture of the gel and rulers (for determining position of
fragment for gel extraction) with UV
- I like to expose the gel to UV for about 2 whole seconds, and
then take a picture with manual exposure set at 0.4 (s?). Waiting a
little bit before taking the picture let's the UV light turn on all
the way and results in a more consistent picture. Note that the
very first time after logging in, turning on the UV light, and
hitting the manual exposure button there is some type of warning
popup so be prepared for that. It might be best to hit manual
exposure first one time before ever turning on the UV light to
first get rid of this warning popup.

o Use image and position information from ruler to cut out the
appropriate bands.
o Result: Gel Extraction Images 082511

o Make sure not to use Ampure kit on vector (only scFv fragment)
o Use Ampure PCR purification
o Keep Ampure PCR Purification treated sample tube on magnetic
plate when transferring solution for other steps

o 9/19/11: We should precipitate with the carrier molecule tRNA
rather than glycogen. Ordered Sigma tRNA (100 U for $35)

o Plate just enough to determine transformation efficiency and
complexity of library
- later on 10-15 of these colonies should be PCR'd to check for
present and size of insert and then sequenced if present.
@ see Colony PCR of Mouse SMC1fs scFv
@ Transformation efficiency of SMC1fs Group: 2*10^7 cfu/ug
@ Transformation efficiency of PCANTAB without insert:
2*10^6 cfu/ug


o Note: I've been having a lot of problems with background colonies
that don't have inserts. See troubleshooting background colonies
without inserts
o I will store the remaining transformation solution in the fridge
to see if an appropriate amount was plated to calculate the
transformation efficiency. If I was off, I can just take some more
volume from this stock in the fridge.
o After the transformation efficiency has been determined, dilute
the transformation culture in LB/carb so that there are 1000 cfu/(1
mL).
o Transfer culture to reagent reservoir (like 25 mL capacity) and
use transfer pipette to transfer 1 mL aliquots into 96 well PCR plate
(2 mL volume 96 well Plate)
o Incubate overnight 37 C 250 rpm
- Note: don't overload PCR plate rack holder in incubator when
it is in a slanted position. Although 10 plates can fit, the holder
is likely to tip over with so many plates. It's probably best just
to put 5 plates in the holder, and tape any extra plates to the
floor. Alternatively, the plate holder can also be screwed to the
floor instead of held in place in a slanted position.
- Note: many of the wells have a tendency to evaporate so it
might be best to keep the overnight incubation on the shorter side
and/or add extra LB to evaporated wells and resuspend.


o combine 1 uL from each culture (all 100) into 1 tube. Add 100 uL
autoclaved glycerol, gently mix by shaking, and store in -80 C

o Transfer culture from PCR wells to reagent reservoir using
multichannel pipette
o Use transfer pipette to transfer 5 mL aliquots to culture tubes
o Pellet cells by centrifuging at about 3,830Xg (5,000 rpm in
Sorvall centrifuge)
- Note: we originally considered using a plate miniprep to
avoid bias arising in the library as the culture grows, but then we
decided against this. See email about miniprep decision


- After miniprep, combine 1 uL of each sample into 1 mixed
tube. This mixed tube can be used for transformation of the helper
ER2738/M13cp-CT Cells)


o Induce protein expression by adding IPTG (final concentration 1
mM). Let protein express 3-4 hours 37 C 250 rpm

o Resuspend pellet in 500 uL LB. Add 500 uL autoclaved glycerol,
gently mix by shaking, and store at -80 C



Questions

o Andrey says it's best to use water if I will use the DNA soon. If
I will take a long time before using the DNA then it is better to use
tris since this can prevent the DNA from degrading

o yes but I can put PCR tubes into the magnetic ampure plate

o Just use about 1-2 uL after about 1 hr.

o He typically uses anywhere between 30-50%. 0.5 mL culture and 0.5
mL some concentration of glycerol

phage?
o 1 mM final IPTG looks reasonable (from Insoluble Protein
Purification from Ecoli. 12/11/06). Then after the culture has been
induced allow the protein to be made for 3-4 hours at 37 C 250 rpm

bacteria up overnight I think right?
o This is correct. I can't make a glycerol stock until the bacteria
is in stationary phase and the bacteria are fully mature with a nice
thick cell wall.

to correctly determine the complexity?
o Yes. But I should also do a colony PCR first to determine the
length of the inserts.

o Now that I think about this more, I think I know the answer. The
complexity is simply the number of unique clones in the library. I
will know this after I sequence some of the colonies.

genes? I think I have seen some people make phage libraries and only
use the kappa genes. see Ratio of Lambda to Kappa Genes
o Kathy and I both think it's okay to only proceed with the kappa
genes since they comprise the majority of the library.


Areas for Improvement After 1st Run Through Protocol with Mouse SMC1fs
Kappa scFv

some won't to test for self ligation)
extract)
when transferring solution for other steps
from uncut vector well and possibly run longer (longer than 3-4 hours).
transformation with PCANTAB-scFv and DH10B cells.
(3830Xg) in large Sorvall centrifuge.
300 uL 96 well plate

Also see notes after presentation



see also
"C:\Users\kurtw_000\Documents\kurt\storage\CIM Research Folder\DR\2014\01-18-2014d1351\wikispaces download 01-18-2014d1351\01-18-2014d1452\Digest - PCANTAB and rCOMP TT with SfiI and NotI with dephosphorylation.xls"