IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-18

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Contents

DNA Miniprep of topo+kaiabc clones

With the Topo prepared clones in e.coli Hetmann created:

 1: kaiabc+topo #1
 2: kaiabc+topo #1
 3: kaiabc+topo #1
 4: kaiabc+topo #1
 5: kaiabc+topo #2
 6: kaiabc+topo #2

I aliquoted 4mL of each topo clone into its seperate vial and conducted a DNA miniprep based on the Qiagen miniprep kit 6/05 handbook p22-23. Used old Qiagen miniprep kit (had very little P2 buffer in it); new one did not have RNAase added. #1,4,and 5 did not have as many cells as 2,3, and 6. Used centrifuge for the large centrifuge tubes; 4.4k for 10 min.

note: tubes 4,5,6 may have been a bit contaminated by either 1,2, or 3; during loading of first centrifuge, but most likely okay.

The final reactions are stored in blue 1.5 tubes marked (number, kaiabc topo DNA). used 30 uL extraction.

DNA nanodrop data:

  1: 153.3 ng/uL
  2: 14.0 ng/uL
  3: 14.4 ng/uL
  4: 8.9 ng/uL
  5: 12.5 ng/uL
  6: 27.2 ng/uL

Weirdly enough, sample 1 and the other ones fell within 260/280 as 1.8-2.0... did NOT dilute sample 1.

Storage of topo+kaiabc clones

For all 6 topo clones, I aliquoted 666uL of each into a cryogenic vial, and added 666uL of 50% glycerol. Each is in a vial at -80C, in a cap with blue writing and dated.

Digest of the topo+kaiabc clones and the one we were using the past week

In order to do a pre-sequence test to see if our template is correct, I did a restriction digest using the pstI enzyme. Based on this Media:Pcr2topoblunt.pdf file, if we cut at the pst1 site only we can do three diagnoses. The following is what I look to test.

    1. If there is no kaiABC in the vector, we will get a 3519 bp fragment.
    2. If there is no kaiABC in the vector and kaiABC freeflowing, we will get a 500 bp fragment, a 2390bp fragment, and a 4519 bp fragment
    3. If there is kaiABC in the vector, we will get a 2390bp fragment and a 4138bp fragment
    4. If there is random crap in the vector, the bands will be different; a 3kb band prehaps?
    5. Positive control: reactions itself?
    6. Negative control: no DNA

Granted, using SpeI or double digesting may provide more info, but I think we're out of SpeI and ... yeah.

Per reactions 2,3,4,5,6 the amount in a 50uL rxn (gives ~140-270ng of DNA/rxn depending on concentration):

  10uL DNA
  5uL 10x Buffer3
  1uL PstI
  0.5uL 100x BSA
  33.5uL dH20

For reaction 1 the amount in a 50uL rxn (gives 300ng of DNA):

  2uL DNA
  5uL 10x Buffer3
  1uL PstI
  0.5uL 100x BSA
  41.5uL dH20

For reaction "Tp" the amount in a 50uL rxn (gives 300ng of DNA, was template we were using last week):

  5uL DNA
  5uL 10x Buffer3
  1uL PstI
  0.5uL 100x BSA
  38.5uL dH20

For negative:

  5uL 10x Buffer3
  1uL PstI
  0.5uL 100x BSA
  43.5uL dH20

Master Mix was preformed for all samples: combining BSA, Buffer3, PstI, and dH20.

The run time is:

 37C for 6 hours
 80C for 20 minutes
 4C for forever

The run is being done on the PCR machine #1. Will be done by 1030AM 7/18.

Repeat of colony PCR (see 7/10)

Assuming that our digests are not turning out good, I repeated the colony PCR step from cyanobacteria. Biggest change here is the use of solid monocolonal colony intstead of liquid polyclonal. Lysing by thermocycler @ 95C @ 5min.

The protocol is:

1 experimental, 1 negative (no template), and 1 BB test.

The experimental/negative had:

  5uL/0uL colony DNA
  1.5uL 20mM dNTP (new)
  1.0 uL extR
  1.0 uL extF
  0.5uL HotStarTaq (N)
  5uL 10x buffer
  41.5/46.5 dH20

The BioBricks test, which looks for contamination in the primers, had:

  1.5uL dNTP
  1.0 uL RF
  1.0 uL (reverse)
  1.0 uL HotStarTaq (N)
  5 uL 10x buffer
  41.5uL dH20

They are in 0.2mL miniPCR tubes in PCR#6. (3 samples total) Tops in blue.

Repeat of 7/17 transformation

I (Jeff) plated yesterday's transformants on the wrong plates, so I redid the Topo ligation and transformation. I used 30 µL, 15 µL, and 15 µL of competent cells for the experimental, negative, and positive controls.

I plated the transformants on 6 plates (1 negative control, 1 positive control, 4 experimental) at around 1 PM.

Gels

Diagnostic digest of old PCR Click for legend
Diagnostic digest of old PCR

Click for legend
New experimental PCR Click for legend
New experimental PCR

Click for legend
Diagnostic digest of old PCR after post-staining Click for legend
Diagnostic digest of old PCR after post-staining

Click for legend
Final run of digest and PCR, @130V@50min, 1%.
Final run of digest and PCR, @130V@50min, 1%.


Primers

We received primers today for sequencing our KaiABC extract and extracting KaiA, KaiB, and KaiC separately. We hydrated the primers and made dilutions as recorded below:

Sequencing primers

Primer Stock concentration Stock volume Dilution concentration Dilution volumes
seqA1179.64 mM250 µL20 mM5.57 µL stock + 44.43 µL H20
seqA2189 mM250 µL20 mM5.29 µL stock + 44.71 µL H20
seqA3205.64 mM250 µL20 mM4.86 µL stock + 45.14 µL H20
seqA4155.2 mM250 µL20 mM6.44 µL stock + 43.56 µL H20
seqA5190.8 mM250 µL20 mM5.24 µL stock + 44.76 µL H20
seqA1_RC201.72 mM250 µL20 mM4.96 µL stock + 45.04 µL H20
seqA2_RC172.88 mM250 µL20 mM5.78 µL stock + 44.22 µL H20
seqA3_RC192.88 mM250 µL20 mM5.18 µL stock + 44.82 µL H20
seqA4_RC218.12 mM250 µL20 mM4.58 µL stock + 45.42 µL H20
seqA5_RC199.4 mM250 µL20 mM5.01 µL stock + 44.98 µL H20

Extraction primers

Primer Stock concentration Stock volume Dilution concentration Dilution volumes
exAF66.52 mM250 µL20 mM15.03 µL stock + 34.97 µL H20
exAR100 mM328.2 µL20 mM10 µL stock + 40 µL H20
exBCF52.52 mM250 µL20 mM19.04 µL stock + 30.96 µL H20
exBCR100 mM309.4 µL20 mM10 µL stock + 40 µL H20
exBOR100 mM165.7 µL20 mM10 µL stock + 40 µL H20
exCOF68.36 mM250 µL20 mM14.63 µL stock + 44.43 µL H20

Reinoculation of the Hetmann Six

We reinoculated Hetmann's 6 plated cultures in 5 mL of LB (w/ 2 µL Kan == 20 µg/mL), using 10 µL liquid culture from the frozen stocks that Peng made in the early morning.
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