Team:Stanford/Notebook/Lab Work/Week 4

From 2010.igem.org

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#RSID2/RBS
#RSID2/RBS
 +
 +
==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)
 +
|}
</div>
</div>

Revision as of 23:51, 21 July 2010

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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 ACL
Need .01 L of 10mM TRIS ACL solution. So, add 15.76 mg TRIS ACL 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


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

  • we'll be using awesome PCR supermix- KEEP ON ICE
  • Add 4 times as much primers when annealing two

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


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)


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