Team:Caltech/Week 13

From 2011.igem.org

(Difference between revisions)
 
(One intermediate revision not shown)
Line 96: Line 96:
Transformed DDT-pET protein overexpression vector into BL21D3 strain<br/>
Transformed DDT-pET protein overexpression vector into BL21D3 strain<br/>
Mix 40mg/mL X-gal (100x) in preparation for X-gal-biofilm experiment<br/>
Mix 40mg/mL X-gal (100x) in preparation for X-gal-biofilm experiment<br/>
 +
PCR amplification for BisdB parts mix using phusion: <br/>
 +
Volume: 50 ul <br/>
 +
* Temp 98°C for 30 seconds
 +
* Temp 98°C for 10 seconds
 +
* Temp 55°C for 20 seconds
 +
* Temp 72°C for 30 seconds
 +
* Go to 2, 9
 +
* Temp 98°C for 10 seconds
 +
* Temp 65°C for 20 seconds
 +
* Temp 72°C for 30 seconds
 +
* Go to 6, 19
 +
* Temp 79°C for 3 minutes
 +
* Temp 4°C forever
 +
* END
===Results===
===Results===
Transformation plating of pSB3k3-bisdB yielded 4 red colonies and one white colony<br/>
Transformation plating of pSB3k3-bisdB yielded 4 red colonies and one white colony<br/>
Line 101: Line 115:
==September 9==
==September 9==
Minipreps and quikchange kit<br/>
Minipreps and quikchange kit<br/>
 +
Quikchange kit pcr for a 25 ul reaction:<br/>
 +
* 2.5 ul of 10x buffer
 +
* 5 ul of plasmid
 +
* 1 ul of primer A
 +
* 1 ul of primer B
 +
* 1 ul of dNTP mix
 +
* 1 ul of lightning
 +
* 0.75 ul of Quik Solution
 +
* 12.75 ul of water
}}
}}

Latest revision as of 18:31, 13 September 2011


Caltech iGEM 2011



Home

Project

Data

Parts

Team

Notebook

Biosafety

Human Impact

References

Support

September 4

Overnights of: K123001 (bisdB) x2, pNT001 (construct vector) x2, and pSB3k3(BioBrick submission vector) x3

September 5

Miniprepped overnights of K123001, pNT001, and pSB3k3
PCR assembly of K123001 with prefix-promoter and terminator-suffix ultrimers to make bisdB construct
Gel of PCR assembly product
Overnights of J23119-lacZ-MG-1655 for x-gal analysis

Results

Miniprep concentrations:

Miniprep Nanodrop conc. (ng/uL)
pNT001-a 41
pNT001-b 83.5
K123001-a 101.8
K123001-b 68.0
pSB3k3-a 69.3
pSB3k3-b 83.4
I372017 0
pSB3k3-c 54.1

Gel reveals correct sized product of ~1.5kbp

September 6

Assembly of bisdB pcr'ed construct into pNT001 (with bisdA) and pSB3k3 (BioBrick submission vector, with unwanted RFP)
Plate all assembled & transformed constructs and controls
Grow overnights of J23119-lacZ-MG-1655 from two different plate sources and one glycerol stock

September 7

Plated the rest of transformed pSB3k3-bisdB
Add the J23119-lacZ-MG-1655 to autoclaved LB+glass beads to incubate biofilm at 37&degC
Performed qualitative X-gal experiment to pick best J23119-lacZ-MG-1655 overnight (turned most blue in least time)
Restreak pNT001-bisdB colonies onto another plate for clearer single colonies

Results

Plate results:

Plate Number of colonies
pNT001-bisdB 15
pSB3k3-bisdB 0
pSB3k3-control #1 (just digestion) 20, all red
pSB3k3-control #2 (digestion + ligation) 6, one white
pSB3k3-control #3 (digest, dephosphorylate, ligate) 0

September 8

Overnights of all pNT001-bisdB and pSB3k3-bisdB colonies for sequencing tomorrow
Transformed DDT-pET protein overexpression vector into BL21D3 strain
Mix 40mg/mL X-gal (100x) in preparation for X-gal-biofilm experiment
PCR amplification for BisdB parts mix using phusion:
Volume: 50 ul

  • Temp 98°C for 30 seconds
  • Temp 98°C for 10 seconds
  • Temp 55°C for 20 seconds
  • Temp 72°C for 30 seconds
  • Go to 2, 9
  • Temp 98°C for 10 seconds
  • Temp 65°C for 20 seconds
  • Temp 72°C for 30 seconds
  • Go to 6, 19
  • Temp 79°C for 3 minutes
  • Temp 4°C forever
  • END

Results

Transformation plating of pSB3k3-bisdB yielded 4 red colonies and one white colony

September 9

Minipreps and quikchange kit
Quikchange kit pcr for a 25 ul reaction:

  • 2.5 ul of 10x buffer
  • 5 ul of plasmid
  • 1 ul of primer A
  • 1 ul of primer B
  • 1 ul of dNTP mix
  • 1 ul of lightning
  • 0.75 ul of Quik Solution
  • 12.75 ul of water


Retrieved from "http://2011.igem.org/Team:Caltech/Week_13"