Team:Stanford/Notebook/Lab Work/Week 4

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Contents

7/19 Monday

Laura's Notebook

set up the following ligations from the gel extractions done by me, Karina, and Francisco on Friday, 7/14/10)

  • Francisco ran and imaged diagnostic gel on Friday
Ligation Recipe
dH2O none
vector (with terminators attached) 5.0 uL
insert (RFP or GFP) 12.0 uL
10X buffer 2.0 uL
T4 ligase 1.0 uL
  • normally run for 10 minutes at room temperature, overnight this time (started at 11:30 am)


Nanodrop data (deemed unreliable based on 260/230, possibly due do residual EtBr contamination)

part 260/280 260/230 ng/uL
vector/terminator 1.77 0.03 19.1
RFP 1.86 0.02 16.2
GFP 1.83 0.02 14.8


Karina's Notebook

Goal: Laura will ligate GFP and RFP and ligate them to terminators. We received our RSID + RBS oligos in the mail, so I will work on the PCR. I first need to make freezer stock and working stock of oligos. Won't start PCR until after lunch because we'll leave them overnight with Chris' PCR reactions.

Make Tris-HCl
Need .01 L of 10mM Tris-HCl solution. So, add 15.76 mg Tris-HCl to 10 mL H20.

  • Hard to weigh out 15.8 mg, so instead got to 18.3 mg.
  • Determined that need to add this to 11.6 mL H20

Make Freezer Stock of Oligos
Want 100 uM solution of Tris-HCl solution

  • Amount of Tris-HCl to add depends on how much of the oligo's we received.
amount (nmol) mass (mg) amount of Tris to add (uL)
RSID 1 + RBS Forward 70.2 1.82 702
RSID 1 + RBS Reverse 76.7 1.85 767
RSID 2 + RBS Forward 81.1 2.22 811
RSID 2 + RBS Reverse 83.5 2.09 835


Make Working Stock
Want a 10uM working stock solution

  • 900 uL water + 100 uL Freezer Stock Solution

PCR Assembly
PCR Protocol calls for:
1.25 uL reverse primer
1.25 uL forward primer
DNA Template
50 uL PCR supermix

  • For PCR Assembly, do not need DNA template. Also, add 4 times as much of each primer for PCR assembly.

Revised Recipe
5 uL Forward Primer
5 uL Reverse Primer
40 uL PCR Supermix

  • we'll be using awesome PCR supermix- KEEP ON ICE
  • Ran PCR with Chris, left running overnight

Francisco's Notebook

  • Helped Karina and Laura

7/20 Tuesday

Laura's Notebook

helped Alex with preparation of competent cells


ran diagnostic gel for PCR-assembled DNA (Karina set up PCR rxns yesterday)

  • order on gel:
  1. 100 bp ladder
  2. RSID1/RBS
  3. RSID2/RBS

Karina's Notebook

Goals: Run gel of PCR Assembly Product, gel extraction, digest, then run PCR clean up.

Make Gel

  • Chris made me an 0.8% agarose gel

Load Gel

  • added 10 uL loading dye directly into PCR tubes
  • loaded 40 uL of each sample into wells

Gel Results

left to right: 1kb ladder, RSID1 PCR reactions A, B and C


File:072010 gel3.jpg
left to right: 1kb ladder, RSID2 PCR reactions A, B and C


Analysis/Observations
Gel looked a little streaky to me, but we really only need the brightest bands (as circled). For next time:

  • use 100 bp ladder (instead of 1 kb)
  • Don't even bother with gel extraction after running a PCR. It is highly inefficient and a lot of DNA will be lost. Instead, run a a diagnostic gel to check that PCR assembly worked but then proceed to run a PCR cleanup and then digest.

  • Gel with 100bp ladder:
left to right: 100bp ladder, RSID 1, RSID 2

Extraction
Extracted 1A, 1B, 2B, and 2C from the gel.

sample tube mass (g) mass w/getl (g) gel mass (g) volume of QG to add (uL)
1A 1.01 1.25 .24 720
1B 1.01 1.28 .27 810
2B 1.01 1.24 .23 690
2C 1.01 1.24 .23 690
  • extracted and incubated each of the samples separately. To combine, loaded 1A and 1B into one spin column and 2B and 2C into another. Loaded up to 800 uL, spun down, loaded, then spun again. Then continued to follow procedure.

PCR Assembly
Set up another PCR assembly of RSID 1 and RSID 2 so that we have samples that have not been gel extracted. Ran another three samples of each. Will let run overnight.

  • Primers were designed to anneal at 55º, but set up a temperature gradient to test annealing at other temperatures.
RSID temperature (ºC)
1A 68
1B 63
1C 56
2A 68
2B 63
2C 56

Greg's Notebook

  • Miniprepped pBAD, F2620, pSB1a2, pSB3c5
  • Ran nanodrop diagnostic:
Part # 280 230 concentration (ng/uL)
pBAD 1.95 1.33 54.7
F2620 1.88 1.48 185.1
pSB3c5 1.71 0.85 40.4
pSB1a2 1.81 1.38 153.4
  • Poured large gel
  • Helped Chris with electroporation transformation

7/21 Wednesday

Laura's Notebook

today's digestions:

  • RSID1, RSID2 (gel extracted from first PCR done by Karina)- digest with XbaI, PstI
    • run overnight; started at 11:30am
  • promoters within backbones (I0500-from Greg's box, and F2620-from Chris' box)- digest with SpeI, then PstI (done by Francisco)
    • SpeI digest run for 3 hours, then heat killed 20 min. at 80oC, then PstI added overnight (to reduce enzyme competition for sites on the DNA, since recognition sequences are very close to each other)

Recipe:

component amount (uL)
DNA 12.0
H2O 26.0
10X NEB buffer #2 5.0
10 X BSA 5.0
each enzyme 1.0 (2.0 total)

Karina's Notebook

Goal: Left Laura to gel extract previous PCR product as I worked on the new PCR product. Today, must make and run a diagnostic gel, PCR clean up, then digest RSID 1 and RSID 2.

Make TAE
Need a 1 L solution; we have 50x TAE stock

  • 20 mL 50x TAE + 980 mL H20

Make Gel
Want a 2% agarose gel because oligos are short (~220 bp).

  • 1 g agarose + 50 mL TAE
  • 10 uL EtBr

Also, made Chris an 0.8% agarose gel (to repay him for yesterday).

Load Gel
10 uL 100 bp ladder
1 uL dye + 1 uL DNA

Results
[link here]

Great! No difference in the annealing temperatures. All bands are clear and bright, can use all three samples for restriction digests.

PCR Cleanup
Used Qiagen MinElute PCR Purification Kit Protocol.

  • 50 uL reaction, therefore use 250 uL buffer PB
  • Followed protocol and eluted in 10 uL water.

Restriction Digest
Cut at X and P

Recipe

  • 10 uL DNA
  • 5 uL NEBuffer 2
  • 5 uL BSA
  • 1 uL Xba
  • 1 uL Pst
  • 28 uL H20

Total volume: 50 uL

Let run overnight in 37º waterbath

Greg's Notebook

  • Ran restriction digest of miniprepped DNA
  • Inoculated 3 tubes each with 5 mL LB and F2620, T9002, pSB3c5, pSB1a2, I0500, E0240
  • Gel-extracted some stuff for Alex
  • Did two electroporations


7/22 Thursday

Laura's Notebook

  • did gel extracion (Qiaquick Gel Extraction Kit) on the following samples:
part/digestion mass tube (g) mass tube + gel (g) mass gel (g) uL QG to add
I0500 (pBAD) 1.00 1.06 0.06 180
F2620 1.00 1.05 0.05 150
RSID1KP 1.00 1.05 0.05 150
RSID1LEO 1.01 1.08 0.07 210
RSID2KP 1.00 1.07 0.07 210
RSID2LEO 1.01 1.05 0.04 120
    • RSID1KP and RSID2KP- PCR done by Karina, then gel extracted prior to digestion, digestion done by Karina
    • RSID1LEO and RSID2LEO- PCR done by Karina, digested by Laura
  • ran on 1.5% gel at 75V for 1 hour
    • 10uL ladder (1KB)
    • 1uL each sample + 1uL loading dye- in order on chart

Made 4 0.5L bottles of agar

  • recipe:
    • 5 g bacto-peptone
    • 2.5 g yeast extract
    • 2.5 g NaCl
    • 6 g agar
    • 485 mL dH2O (did first bottle in graduated cylinder, powders shifted volume by 15 mL- 450 mL went up to 465 mL, mixed and added to bottle, then used 35 mL dH20 to rinse cylinder contents into bottle)


7/23 Friday

Laura's Notebook

designed primers for colony PCR

tips for primer design (from Smolke Lab, via Chris VanLang)

  • 18-24 bases
  • ~60oC annealing temperature
  • avoid: high or low GC content, repeats of >3 bases
  • for use in later sequencing reactions, place primers ~ 50 bp outside region of interest

ApE files, with primer sequences annotated:


pairs:

  • pSB2K3, I0500 (pBAD)
  • pSB1A2, F2620 (pLUX)