Team:Stanford/Notebook/Lab Work/Week 7

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8/9 Monday

Francisco's Notebook

GFP, RFP - terminator ligations

Promoter - RSID, sRNA ligations

• Retransformed pLux ligations
• Tried using 1 uL of DNA this time.
• pLux + RSID2: time constant 4.4 ms, 1786 volts
• pLux + sRNA1: 4.3 ms, 1786 volts*
• pLux + sRNA1c: 4.4 ms, 1786 volts
  • second try. first try sparked, TC = 2.5 ms

• Incubated in SOC in the rotisserie for an hour, spun down at 5000g, 3 min, took out 750 uL, plated remaining (~130 uL) overnight

• edit: no colonies =( =( =(

Laura's Notebook

• helped Alex NanoDrop some of his samples (concentrations also written on tops of tubes)

	260/280	260/230	ng/uL
sGFP mDNA	1.71	1.01	320.3
sGFP mDNA s	1.91	1.41	220.7
F2620	1.10	0.87	45.0
F2620 s	1.52	0.88	53.1
I5	1.90	1.06	165.8
I5s	1.98	1.22	113.9
I4	1.73	0.95	384.2
I0500	1.86	0.89	67.7
C1	1.77	0.80	83.0
C2	1.83	0.83	86.6
C3	1.95	1.09	75.7

Karina's Notebook

To 3A or not to 3A... that is the question.

Amount of DNA in ligation is key!

• For 10 uL ligation, never put less than 150 ng of backbone
  
  

Running low on stock of RSIDs and sRNAs, need to make more in order to proceed with assembly.
Also need to digest pBad and pLux

Make Antibiotic Plates
Recipe (per 500 mL bottle)
5 g Bacto-Peptone
2.5 g Yeast Extract
2.5 g NaCl
6 g Agar

• measure out and add to each bottle. Add 450 mL millipore H20 and shake vigorously.
• Total Volume --> 500 mL
• Adhere autoclave tabe, LOOSEN CAP, and set in autoclave tray
• Start program (autoclave liquid)
• Once program ends, use GLOVES to remove bottles and let cool. Once cool enough to keep your hand on the bottle for at least 10 s, add antibiotic.
  
  

Antibiotic Concentrations:
A 50 ug/mL
K 20 ug/mL
C 34 ug/mL
T 20 ug/mL

From a 1000x stock, add .5 mL antibiotic to 500 mL solution. Pour plates and let solidify over night.

Sequencing Analysis GFP looks good! RFP has wierd, additional bases at the very end of RFP but before suffix. It could be part of the BioBrick part, will send E1010 for sequencing tomorrow just to double check.

8/10 Tuesday

Francisco's Notebook

GFP, RFP - terminator ligations

Promoter - RSID, sRNA ligations

• Decided to try 3A assembly.

PCR assembly of RSID’s and sRNA’s

• Doubled DNA volume: 10 uL fwd piece, 10 uL rev piece, 30 uL PCR supermix
  • total: 50 uL reaction volume
• Set up 3 reaction tubes each for RSID1, RSID2, sRNA1, sRNA2, sRNA1c, sRNA2c.
• Ran FLK program (see above)

Inoculate pLux, pBad

• 2 tubes for each promoter, 6 mL of culture.

Laura's Notebook

set up colony PCR for single colony from pLUX-sRNA1C ligation/transformation:

• 9 uL supermix, 0.5 uL of each primer (VF, VR)
• cycling program as before: COLPCR72

3pm meeting with Drew, Rayka about device names, parameters

Karina's Notebook

Goal: 3A Assembly

We don't have enough DNA, need to redo PCR assembly. Upscaling:
10 uL of each primer
30 uL supermix

3 reactions of each product. 10 cycle PCR.

8/11 Wednesday

Francisco's Notebook

GFP, RFP - terminator ligations

Sent in miniprepped RFP (E1010) for sequencing

• There was a funny sequence in the RFP-term ligations. This is to check if that sequence is part of the RFP BioBrick part.

Inoculated G1, G3, R1, R3 for freezer stock and miniprep (Karina)

Promoter - RSID, sRNA ligations

• Miniprep pBad, pLux (Laura)
• Eluted in 50 uL nuclease-free water
• Nanodrop results: 260/280, 260/230, ng/uL
  • pBad: 1.93, 1.53, 112.0
  • pLux: 2.00, 1.67, 149.8

Diagnostic Gel of RSID’s and sRNA’s

• Combined three 50 uL PCR reactions into one volume.
• 2% gel, 95 V, 30 minutes, 1 uL of sample each
  • left to right: RSID1, RSID2, sRNA1, 100 bp ladder, sRNA1c, sRNA2c

• No distinct bands, smearing above expected length, nothing below expected length (so no left-over starting pieces?)
  • Note, this was the PCR assembly done with twice as much DNA as previous PCR’s

Retry PCR Assembly of RSID’s and sRNA’s (vary DNA amounts)

• Troubleshooting PCR assembly. Used only RSID2 and sRNA1c, and tried different amounts of DNA:
  • A: 0.25 fwd, 0.25 rev, 9.5 PCR supermix
  • B: 0.50 fwd, 0.50 rev, 9.0 PCR supermix
  • C: 1.00 fwd, 1.00 rev, 8.0 PCR supermix
  • D: 2.00 fwd, 2.00 rev, 6.0 PCR supermix
  • (PCR tube labeling: R = RSID2, S = sRNA1c)

Diagnostic Gel of PCR Assembly (with varied DNA amounts)

• Results not great, best result with ratio C (see above), but still smeary.

Retry PCR Assembly (vary annealing temperature)

• Tried 50, 55 and 60 deg C

Inoculated pLux-sRNA1c ligation, possibly for sequencing (Karina)

• edit: colony PCR negative, no sequencing =(

Other

Prepared working stock of 1 kb ladder

• 440 uL water
• 100 uL 6x loading dye
• 60 uL 1 kb ladder
• total: 600 uL total volume
• (Diluted so that 10 uL of working ladder contains 0.5 ug of DNA)

Laura's Notebook

miniprepped pBAD, pLUX

• used Promega kit
• combined 2 liquid cultures for each into 1 tube (when pelleting cells)
• eluted in 50 uL H2O (from kit)

set up colony PCR (same as yesterday; tube dried up in machine)

troubleshooting PCR stitching reactions (prior to ligations)- recommendations from Christina:

• each "step:" try RSID2, sRNA1C; use best result to continue to next "step"

1. different starting DNA concentrations: 0.25X, 0.5X, 1X, 2X
2. different annealing temperatures: 50oC, 55oC, 60oC
3. cycle numbers: 5, 10, 15, 20

Step 1: try different DNA concentrations

• Francisco set up tubes and added PCR SuperMix (10 uL reactions)
• I added primers to 1 series of 4, he did the other
• thermal cylcler program: FLK
• run on 2% gel at 90V for 30 minutes

Step 2: try different annealing temperatures

• use 1 uL each primer; 8 uL PCR SuperMix in each reaction (3 + 3 + 24 in one tube, then 10 uL aliquoted to other 2 tubes)
• cycling (FLK program) modified to include a gradient at the annealing step: 50oC to 62oC
  • row H = 50oC, row E = 54.8oC, row C = 59.6oC

Karina's Notebook

Goal: Send RFP in for sequencing, set up liquid culture of RFP + T and GFP + T to make freezer stocks tomorrow.

Sequencing
Need to send Ryan:

• Name of plasmid (RFP)
• size of plasmid (5106, includes plasmid and RFP insert)
• Concentration (179.8 ng/uL)
• primers to use (VF2)
  
  

To determine what primers to use, check plasmid map on parts registry. Plasmid mush have VF regions.
Prep 10 uL of sample in one tube and 3uMol Primer in another. Tape to sheet and place in envelope outside door to send off.

Inoculate RFP + T and GFP +T
After analyzing sequencing results, saw that G1 and R3 colonies have a single point mutation. Keep this in mind, will still inoculate but will only use G3 and R1 for further cloning.

8/12 Thursday

Laura's notebook

ran gel with yesterday's PCR stitching reactions (2%, 90 V, 30 minutes)

• 50oC sample for sRNA1C has disappeared- only 55oC and 60oC samples

Miniprepped RFP-term and GFP-term

• Promega kit used
• 2 liquid cultures of each one, combined into one prep for each
• eluted in 50 uL H2O (provided with kit)

8/13 Friday

Laura's Notebook

tried Chris VanLang's suggestion for PCR stitching (for RSID2, sRNA1C): 1 cycle, ramp from denaturing to annealing at 1oC/sec

• reaction mix: 8 uL PCR SuperMix, 1 uL each primer
• cycling:

94oC	5 minutes
ramp to 55oC at 1oC per second
55 oC	1 minute
72oC	15 seconds

• ran 5 uL of each sample on 2% gel at 90V for 30 minutes (along with 5uL each sample from yesterday (10 cycles, different annealing temperatures)

Next step (Christina's suggestions): different numbers of cycles, extend for 15 seconds (again, only with RSID2 and sRNA1C for now) Cycling program:

step	temperaure	time
1- initial denaturing	94 oC	2 min.
2- denature	94oC	30 sec.
3- anneal	55 oC	30 sec.
4- extend	72 oC	15 sec
repeat steps 2-4 two, 4, 6 or 9 times (total of 3, 5, 7 or 10 cycles)
5- final extension	72oC	10 min.