SBB12Ntbk-JonWright

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Jon Wright 3/22/2012

Digested AmilCP part. Ran gel. Lane 8 below (key on the main page is wrong).

Image:2012_03_22_gel1_ssb2012spring.jpg‎

Appears that previous step was not in fact the desired product. GG.

Jon Wright 3/20/2012

Executed analytical digest on KILD (Bam and Eco).

Ran gel on Miniprep and JWA6-2 (most recent AmilCP product). Lanes 3, 7, and 10 below.

Image:2012_03_20_gel2_ssb2012spring.jpg‎

KILD lane empty. AmilCP lanes MIGHT contain correct part. Proceeded on assumption that they did, due to lack of time to repeat steps.

Jon Wright 3/15/2012

Ran gel on all AmilCP PCR tubes to check that they contained what I thought they contained. All JWs below.

Image:2012_03_15_gel1_ssb2012spring.jpg

Determined that JWA5-1 did contain the correct band and that previous gels had been read incorrectly. Began setting up PCR to amplify, but in the process screwed up another Zymo and washed out the only A5-1 I had left. Set up another PCR2 on previous step.

Jon Wright 3/13/2012

Ran Zymo on PCA1 products for KILD. Set up KILD PCA2.

Made note to design analytical digest for the one successful KILD miniprep.

Ran gel on AmilCP PCR product. Lane 7 below.

Image:2012_03_13_gel2_ssb2012spring.jpg


Jon Wright 3/8/2012

Ran gel on the pre-PCR2 AmilCP parts to double-check contents. Lanes 4 and 7 below.

Image:2012_03_08_gel1_ssb2012spring.jpg‎

Fragments appeared to be correct. Set up second PCR again.

Cleaned up KILD PCA1 products. Screwed up Zymo procedure. Attempted to salvage and re-cleaned. Screwed up again. Cursed loudly.

Set up PCA1 for KILD.

Jon Wright 3/6/2012

Down with the sickness.

("The sickness" here being some form of flu)

Jon Wright 3/2/2012

Ran gel on AmilCP PCRproduct. Lane 9 below.

Image:2012_03_02_gel1_ssb2012spring.jpg

Set up digestion on AmilCP parts while gel was running. Discarded - PCR failed.

Finally got a white colony of KILD. Miniprepped, then ran out of time.

Jon Wright 3/1/2012

Ran gel on AmilCP PCRs. Lanes JW A1 and A2 below:

Image:2012_03_1_gel1_ssb2012spring.jpg‎

Small Frag Zymo purification on AmilCP PCRs.

Set up PCR of ca998/g00101 on combined AmilCP PCRs

Checked KILD colonies; nothing grew. Picked a 3rd round.

Restarted KILD. PCA1 on o1, o2, o9.

Jon Wright 2/28/2012

Started miniprep on KILD. Colonies were all "secretly" red. Re-picked.

Ran gel on AmilCP. Lanes were completely empty. "JWA" below.

Image:Gel1_from_class_2_28_2012.jpg‎

Restarted AmilCP. Set up PCRs: ca998/jw000R on jtk2801 jw000F/g00101 on jtk3346

Jon Wright 2/24/2012

Regular Zymo on AmilCP PCR2

Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction.
Transfer into the Zymo column (small clear guys)
spin through, discard waste.
Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
spin through, discard waste.
Add 200 uL of Zymo Wash Buffer
spin through, discard waste.
spin for 90 seconds, full speed to dry.
elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction

Picked 4 large colonies from KILD plate and inserted into media

For each construct you will pick and later miniprep 2 colonies
Add 4mL of LB media with the appropriate antibiotics to a clean test tube
Pick a well-isolated, round, and "normal" looking colony with a toothpick
Drop it in the test tube
Incubate at 37 overnight

OpenWetWare crashed shortly after I started the Zymo; without physical copies of the protocols (I knew I should have printed them out, urgh), no further progress was possible.

Jon Wright 2/23/2012

Set up SOE PCR on AmilCP subparts (gel-purified PCR products)

24uL ddH2O
3.3uL 10x Expand Buffer "2"
3.3uL dNTPs (2mM in each)
1uL ca998, 10uM
1uL g00101, 10uM
0.5uL Expand polymerase "1"
0.5uL Subparts

Small Frag Xymo on KILD (the PCA product)

Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
Transfer into the Zymo column (small clear guys)
Add 500uL of Ethanol and pipette up and down to mix
spin through (15s), discard waste.
Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
spin through (15s), discard waste.
Add 200 uL of Zymo Wash Buffer
spin through, discard waste.
spin for 90 seconds, full speed to dry.
elute with water (spin 60s) into a fresh Eppendorf tube

Ligation on KILD and plasmid 1834

 6.5uL ddH2O
 1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
 1uL Plasmid 1834 digest
 1uL KILD digest
 0.5uL T4 DNA Ligase

Transformed KILD into chassis and plated on Spec

Thaw a 200 uL aliquot of cells on ice
  2. Add 50 uL of water to the cells (if greater volume is desired)
  3. Add 30 uL of KCM to the cells 
  4. Put your ligation mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution)
  5. Add 70 uL of the cell cocktail to the ligation, stir to mix
  6. Let sit on ice for 10 min
  7. Heat shock for 90 seconds at 42 (longer incubation may work better)
  8. Put back on ice for 1 min
  9. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour
 10. Plate 70+ uL on selective antibiotics, let incubate at 37 degrees overnight


Jon Wright 2/21/2012

Set up and ran gel on PCA2. Lane 8 below:

Image:2012_02_21_gel1_ssb2012spring.jpg

Small Frag Xymo on PCA2

Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
Transfer into the Zymo column (small clear guys)
Add 500uL of Ethanol and pipette up and down to mix
spin through (15s), discard waste.
Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
spin through (15s), discard waste.
Add 200 uL of Zymo Wash Buffer
spin through, discard waste.
spin for 90 seconds, full speed to dry.
elute with water (spin 60s) into a fresh Eppendorf tube

Digested PCA2 with Nhe, Bam

 50uL eluted DNA
 5.7uL NEB Buffer 2
 0.5uL NheI
 0.5uL BamHI

Small Frag Xymo on the gel purification product from last time (since the promoter part is considered a small one).

Jon Wright 2/17/2012

Small Frag Xymo on PCA product

Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
Transfer into the Zymo column (small clear guys)
Add 500uL of Ethanol and pipette up and down to mix
spin through (15s), discard waste.
Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
spin through (15s), discard waste.
Add 200 uL of Zymo Wash Buffer
spin through, discard waste.
spin for 90 seconds, full speed to dry.
elute with water (spin 60s) into a fresh Eppendorf tube

PCA2 on PCA product

1 ul o1
1 ul o2
1 ul PCA1
.5 ul phusion
10 ul 5x phusion buffer
5 ul 2mM dNTPs
32.5 ul H2O

Small Frag Xymo on ca998/jw000R product (see above) Regular Xymo on G00101/jw000F product

Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction.
Transfer into the Zymo column (small clear guys)
spin through, discard waste.
Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
spin through, discard waste.
Add 200 uL of Zymo Wash Buffer
spin through, discard waste.
spin for 90 seconds, full speed to dry.
elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction

Ran gel on ca998/jw000R and G00101/jw000F - lanes 1 and 2, below

Image:NEB_2-log_ladder.gif

Cut bands out of Gel and suspended together in ADB Buffer

Jon Wright 2/16/2012

Diluted JW000R and JW000F to 10uM

Set up PCRs:

24uL ddH2O
3.3uL 10x Expand Buffer "2"
3.3uL dNTPs (2mM in each)
1uL ca998, 10uM
1uL jw000R, 10uM
0.5uL Expand polymerase "1"
0.5uL Plasmid jtk2081
24uL ddH2O
3.3uL 10x Expand Buffer "2"
3.3uL dNTPs (2mM in each)
1uL G00101, 10uM
1uL JW000F, 10uM
0.5uL Expand polymerase "1"
0.5uL Plasmid 3346

Set up PCA:

38 uL ddH2O
5 ul 10x expand buffer
5 ul 2mM dNTPs
0.33 ul o1
0.33 ul o2
o.33 ul o9
0.75 ul Expand polymerase
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