Team:Stanford/Notebook/Lab Work/Week 4

From 2010.igem.org

(Difference between revisions)
(Karina's Notebook)
(Karina's Notebook)
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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. <br/><br/>
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. <br/><br/>
-
'''Make TRIS ACL'''<br/>
+
'''Make Tris-HCl'''<br/>
-
Need .01 L of  10mM TRIS ACL solution. So, add 15.76 mg TRIS ACL to 10 mL H20.
+
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.
*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 <br/>
*Determined that need to add this to 11.6 mL H20 <br/>
'''Make Freezer Stock of Oligos'''<br/>
'''Make Freezer Stock of Oligos'''<br/>
-
Want 100 uM solution of TRIS ACL solution <br/>
+
Want 100 uM solution of Tris-HCl solution <br/>
-
*Amount of TRIS ACL to add depends on  how much of the oligo's we received.  
+
*Amount of Tris-HCl to add depends on  how much of the oligo's we received.  
{| border="1"
{| border="1"
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|amount (nmol)
|amount (nmol)
|mass (mg)
|mass (mg)
-
|amount of TRIS to add (uL)
+
|amount of Tris to add (uL)
|-
|-
|RSID 1 + RBS Forward
|RSID 1 + RBS Forward

Revision as of 17:38, 22 July 2010

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 Recipe calls for:
1.25 uL reverse primer
1.25 uL forward primer
50 uL PCR supermix

  • 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

7/20 Tuesday

Laura's Notebook

helped Alex with preparation of competent cells

  • see protocol: [http://openwetware.org/wiki/Stanford/BIOE44:Module_1:Day3 Preparing Electrocompetent 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
[insert picture here]

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.

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
    • 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)