Team:Stanford/Notebook/Lab Work/Week 4

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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

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.
  
  

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 [http://molecool.wustl.edu/krolllab/Kroll_Lab_Protocols/Molecular%20Biology%20protocols/Cloning%20protocols%20folder/Gel%20extraction-Qiagen.pdf procedure].
  
  

PCR Assembly

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)