Team:Stanford/Notebook/Lab Work/Week 6

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(Difference between revisions)
(→Greg's Notebook)
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** *There wasn't enough pBAD, so I used all that was left (~20uL). Even if this digest fails I still have the previously-digested stuff at a low-ish concentration
** *There wasn't enough pBAD, so I used all that was left (~20uL). Even if this digest fails I still have the previously-digested stuff at a low-ish concentration
 +
=== Laura's Notebook ===
 +
NanoDrop data for Alex's miniprep samples:
 +
{|
 +
| || 260/280 || 260/230 || ng/uL
 +
|-
 +
| I13500 || 1.82 || 1.31 || 221.7
 +
|-
 +
| J23106 || 1.85 || 1.42 || 187.6
 +
|-
 +
| J23109 || 1.88 || 1.47 210.5
 +
|}
 +
 +
Suggestions from Christina:
 +
 +
#diagnostic gels from yesterday's digestions (done by Francisco)
 +
*run promoters (pBAD, pLUX) on 0.8%, RSID's (1 and 2) and sRNA's (1, 2, 1C, 2C) on 2%
 +
*gel extract everything (on separate gels), depending on how complete digestions look- if PCR cleanup, SIP treat
 +
#PCR cleanup inserts (PCR assembled parts)
 +
*use MiniElute columns
 +
*use 10 mM Tris instead of H2O (water here is acidic, and elution is pH-dependant)
 +
*let sit 1 minute before eluting
 +
*load 1 uL on diagnostic gel
 +
#check enzymes to see if getting low: order more if necessary
 +
#colony PCR
 +
*primers: complementary to prefix and suffix or VF and VR? (VF and VR- further from ends to allow subsequent sequencing)
 +
**expected lengths with and without insert: ladder choice, gel %, extension time
 +
*10 uL reactions, run 5 uL on gel
 +
*setting up reactions:
 +
**make, aliquot master mix (SuperMix + primers)
 +
**pick colony with innoculating needle: inoculate PCR reaction, then streak on small square or wedge on a plate
 +
**
== 8/5 Thursday ==
== 8/5 Thursday ==

Revision as of 21:36, 4 August 2010

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Contents

8/2 Monday

Greg's Notebook

  • Inoculated freezer stocks of most of our parts with Alex
  • Began planning for promoter characterization project:
    • Going to do 3A ligations of the form: promoter + superfolded GFP + Kan plasmid
    • Process:
      • Digest parts with at least 2 ug DNA
      • PCR cleanup
      • Diagnostic gel (remember to save uncut DNA for control)
      • Ligate in PCR tubes
      • Heat inactivate at 65 C for 20 min
  • Performed first digestion (F2620 + sfGFP + pSB4k5)
Part # Part amount BSA NEB (#, amount) Enzymes (1 uL each) Water
F2620 10.8 3 2, 3 E + S 11.2
pSB4k5 20 3 3, 3 E + P 2
sfGFP 11.76 3 2, 3 X + P 10.24
  • Let digestions run for 1 hour at 37 C
  • Ran diagnostic gel:
    • sfGFP digested nicely, F2620 yielded 3 bands corresponding to insert, cut plasmid, and uncut plamid
    • pSB4k5 gave only one band and a smudge, so I decided to redigest with only .5 uL of each enzyme, leaving it to run overnight


8/3 Tuesday

Greg's Notebook

  • Ran diagnostic gel of overnight pSB4k5 digest
    • Still didn't work; there were 3 bands, the smallest of which was about the size of the plasmid backbone, and there was no band corresponding to the ccdB insert
  • Decided to re-redigest pSB4k5 using .5 uL of each enzyme for only 1 hour
    • After running on a diagnostic gel, discovered that this, too, failed in the same way as the previous digestions had. I decided to try other Kan plasmids
  • PCR'd Chris's transcription factor promoter (pTFc)
  • Ran nanodrop of pTFc and yesterday's sfGFP and F2620:
Part # 230 280 Concentration
pTFc A 1.36 1.79 196.1
pTFc B 2.08 1.86 159.7
sfGFP 1.89 1.87 52.9
F2620 1.82 1.82 35.5


8/4 Wednesday

Greg's Notebook

  • Performed restriction digest of pTFc, J23100, J23106, J23109, F2620, pSB2k3, pBAD*, pSB1k3
    • *There wasn't enough pBAD, so I used all that was left (~20uL). Even if this digest fails I still have the previously-digested stuff at a low-ish concentration

Laura's Notebook

NanoDrop data for Alex's miniprep samples:

260/280 260/230 ng/uL
I13500 1.82 1.31 221.7
J23106 1.85 1.42 187.6
J23109 1.88 1.47 210.5

Suggestions from Christina:

  1. diagnostic gels from yesterday's digestions (done by Francisco)
  • run promoters (pBAD, pLUX) on 0.8%, RSID's (1 and 2) and sRNA's (1, 2, 1C, 2C) on 2%
  • gel extract everything (on separate gels), depending on how complete digestions look- if PCR cleanup, SIP treat
  1. PCR cleanup inserts (PCR assembled parts)
  • use MiniElute columns
  • use 10 mM Tris instead of H2O (water here is acidic, and elution is pH-dependant)
  • let sit 1 minute before eluting
  • load 1 uL on diagnostic gel
  1. check enzymes to see if getting low: order more if necessary
  2. colony PCR
  • primers: complementary to prefix and suffix or VF and VR? (VF and VR- further from ends to allow subsequent sequencing)
    • expected lengths with and without insert: ladder choice, gel %, extension time
  • 10 uL reactions, run 5 uL on gel
  • setting up reactions:
    • make, aliquot master mix (SuperMix + primers)
    • pick colony with innoculating needle: inoculate PCR reaction, then streak on small square or wedge on a plate

8/5 Thursday

8/6 Friday

Retrieved from "http://2010.igem.org/Team:Stanford/Notebook/Lab_Work/Week_6"