Plan for Cloning Mouse scFv DNA 082411

2015-01-13

azim58 - 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

* PCANTAB5E(1 ug)
* PCANTAB5E Single Cut
* PCANTAB5E Uncut
* SMC1fs Kappa scFv (all that I have; probably about 100 ng)
  o see Final scFv DNA 11xx10

Protocol Outline

* Restriction Digests
  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.

* Treat vector with SAP(shrimp alkaline phosphatase)
  o Our SAP is from Promega

* Gel Extract vector
  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

* PCR Purify scFv Fragment
  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

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

* Ethanol Precipitation
* Transformation/Electroporation(DH10B Commercially Competent Cells)
  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.

* Glycerol Stock
  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

* Miniprep
  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)

* Prepare fresh electrocompetent cells with "helper plasmid" M13cp-CT.
* Transformation/Electroporation(ER2738/M13cp-CT Helper Cells)
  o Induce protein expression by adding IPTG (final concentration 1
    mM). Let protein express 3-4 hours 37 C 250 rpm

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

* Titrate

Questions

* elute with water or tris-acetate (pH 8) or TE with Ampure?
  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

* have to use 96 well plate with ampure right?
  o yes but I can put PCR tubes into the magnetic ampure plate

* how much ethidium bromide should be added to bottom part of gel?
  o Just use about 1-2 uL after about 1 hr.

* 50 or 80% glycerol stock?
  o He typically uses anywhere between 30-50%. 0.5 mL culture and 0.5
    mL some concentration of glycerol

* What concentration IPTG should I use to induce the production of the
  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

* I won't be able to make a glycerol stock until after I grow the
  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.

* Should I sequence some of the colonies from the first transformation
  to correctly determine the complexity?
  o Yes. But I should also do a colony PCR first to determine the
    length of the inserts.

* What are the units of complexity?
  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.

* Is it very necessary to process the lambda genes as well as the kappa
  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.

* How long can transformation solution be stored in fridge?

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

* Treat all vector samples with SAP (some will just have insert and
  some won't to test for self ligation)
* Don't treat cut vector with Ampure PCR Purification kit (just gel
  extract)
* Keep Ampure PCR Purification treated sample tube on magnetic plate
  when transferring solution for other steps
* Use a little bit lower % agarose (0.8 instead of 1) to separate cut
  from uncut vector well and possibly run longer (longer than 3-4 hours).
* Add antibiotic (ampicillin or Carb) to overnight culture after 1st
  transformation with PCANTAB-scFv and DH10B cells.
* Don't centrifuge culture tubes at 16.1 rcf. Just do 5,000 rpm
  (3830Xg) in large Sorvall centrifuge.
* Make sure to make glycerol stock before pelleting cells for miniprep
* Better to grow cultures in 2 mL volume 96 well Plate instead of in
  300 uL 96 well plate

Also see notes after presentation

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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"

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