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Team:GeorgiaTech/WeekThirteen
From 2010.igem.org
10/24/2010
Plated RFP expression studies begun:
10/25/2010
Goals
- image hyb.ompa.rfp-f3r colony R11 plates to check for periplasmic expression (COMPLETED!)
- obtain SpeI from Hammer Lab (COMPLETED)
- digest A5 miniprep (10.23, in yellow rack) with EcoRI and SpeI (COMPLETED)
- make more gels
- this afternoon, gel extract the 1.5 kb isnert from A5 on a gel
- upload pictures from periplasmic expression to iGEM NB - send out an e-mail to all team members if possible to show these so that we can use the images for slides and poster.
Protocols
Digest of colony A5 of hyb.ompa.aoxA.psb1a3 (from 10.23.2010)(Christina)
colony 5, Aox1a colony PCR from 10-22 (117 ng/ul)
4.5 uL H20 -
2uL 10X EcoRI buffer -
10 uL hyb.ompa.aoxa.psb1a3 colony 5 miniprep (10.22.2010) -
2 uL BSA (1ug/uL, to a final conc of .1mg/ml) -
0.75 uL SpeI-
0.75 uL EcoRI-
Total=20 ul total
put on heat block at: 10:40 am
Imaging of the hyb.ompa.rfp-f3r R11 constructs
Condition | Filename | Observations | Slide Preparation |
|
Condition 8 (RT, 23c) | iGEM_1 | Periplasmic expression | PBS, L-Lysine slide |
|
| iGEM_2 |
| - |
|
| iGEM_3 |
| - |
|
| iGEM_4 |
| - |
|
Condition 7 (RT, transferred to 37c) | iGEM_5 | periplasmic expression but dimmer than 8 | - |
|
Control (from 10.16 plate)(biobrick vector) | iGEM_6 | cells are red throughout, appearing as solid ellipse | - |
|
| iGEM_7 | cells are red throughout, appearing as solid ellipse | - |
|
Cond 5 (20c, transferred to 37c) | iGEM_8 | periplasmic expression | - | Same gain as iGEM-9 |
Cond 6 (20c) | iGEM_9 | periplasmic expression | - | same gain as iGEM-8 |
| iGEM_10 | periplasmic expression | - | increase gain |
Gel for gel extraction
order: gene exact ladder|digested psb1c3|digested A5 (10.23)|ladder|empty|digested psb1c3|digested A5 (from 10.23)
started 5:20 pm
(before gel extraction)
After gel extraction
Notes: the predicted 1.5 kb band was excised from the colony A5 hyb.ompa.aoxa digest, but the digest of the vector did not yield the predicted 2 kb backbone.
Need to report the digest of psb1c3 with EcoRI and SpeI
hyb.ompa.aoxa A5 SpeI and EcoRI digest (2 kb band) gel extraction (Scott,Debika)
1. Excised DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weighed the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg or approximately 100 μL).
Weight= 190 mg
3. Incubated at 50ºC for 10 min (or until the gel slice had completely dissolved). To help dissolve gel, mixed by vortexing the tube every 2 – 3 min during the incubation.
4. After the gel slice has completely dissolved, checked that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).
5. Added 1 gel volume of isopropanol to the sample and mixed.
6. Placed a QIAquick spin column in a provided 2 mL collection tube.
7. To bind DNA, applied the sample to the QIAquick column, and centrifuged for 1 min @13,000k.
8. Discarded flow-through and placed QIAquick column back in the same collection tube.
9 To wash, added 0.75 mL of Buffer PE to QIAquick column and centrifuged for 1 min @13,000.
10. Discarded the flow-through and centrifuged the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm).
11. Placeed QIAQuick column into a clean 1.5 mL microcentrifuge tube.
12. To elute DNA, added 30 μL water (pH 7.0 – 8.5), let the column stand for 1 min, and then centrifuged for 1 min @13,000k.
RE Double Digest Recipe for pSB1C3 miniprep (from 10/19/2010) -- digest with speI and EcorI (Christina, Scott)
12.5 uL H20 -
3uL EcoRI Buffer -
10 uL pSB1C3 (10.19.2010, 106 ng/uL)-
3 uL BSA (1ug/uL, to a final conc of .1mg/ml) -
0.75 uL SpeI-
0.75 uL EcoRI-
Total=30 ul total
Digest overnight - put in waterbath 37 degrees at 7:12 pm
Predict a insert (1000 bp) and backbone (2kb)
Nanospec: 27 ng/uL
gel for gel extraction:
ladder (exactgene)|4uL psb1c3|10 uL digested psb1c3 (10.25.2010)|10 uL digested psb1c3 (10.25.2010)
(before gel extract)
(after gel extract)
psb1c3 (miniprep from 10.19.2010) SpeI and EcoRI digest (2 kb band) gel extraction (Repetition of the digest) (Christina, Scott)
1. Excised DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weighed the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg or approximately 100 μL).
Weight= 290 mg
3. Incubated at 50ºC for 10 min (or until the gel slice had completely dissolved). To help dissolve gel, mixed by vortexing the tube every 2 – 3 min during the incubation.
4. After the gel slice has completely dissolved, checked that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).
5. Added 1 gel volume of isopropanol to the sample and mixed.
6. Placed a QIAquick spin column in a provided 2 mL collection tube.
7. To bind DNA, applied the sample to the QIAquick column, and centrifuged for 1 min @13,000k.
8. Discarded flow-through and placed QIAquick column back in the same collection tube.
9. Recommended: Added 0.5 mL of Buffer GQ to QIAquick column and centrifuged for 1 min.
10. To wash, added 0.75 mL of Buffer PE to QIAquick column and centrifuged for 1 min @13,000.
11. Discarded the flow-through and centrifuged the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm).
12. Placeed QIAQuick column into a clean 1.5 mL microcentrifuge tube.
13. To elute DNA, added 30 μL water (pH 7.0 – 8.5), let the column stand for 1 min, and then centrifuged for 1 min @13,000k.
Ligation of psb1c3 gel extraction (2 kb backbone) to gel extraction of hyb.ompa.aoxa colony A5 insert (1.5kb insert)
use 2:1 product:vector
Ligation:
2 uL of insert (gel extraction from colony A5) 1.5 kb = 0.0054 equivalents/uL -
1.7 uL of vector (2kb backbone from gel extraction of psb1c3) 2 kb 7.1 ng/ul = 0.0035 equivalents/uL-
1 uL 10x T4 Ligase Buffer -
4.8 uL H20 -
0.5 uL T4 Ligase
Total=10 uL
leave at RT for 2 hours
start 11:43 pm
Started: 11:15am 10/26/10
Heat shock transformation of the psb1c3+hyb.ompa.aoxa
10 սL BL21 cells + 5 սL of ligation reaction
(note, psb1c3 is choloamphenicol resistant!)
See Protocols Page for Heat Shock Transformation
10/26/2010
Ligation of psb1c3 gel extraction (2 kb backbone) to gel extraction of hyb.ompa.aoxa colony A5 insert (1.5kb insert) (Christian)
use 2:1 product:vector
Ligation:
2 uL of insert (gel extraction from colony A5) 1.5 kb = 0.0054 equivalents/uL -
1.7 uL of vector (2kb backbone from gel extraction of psb1c3) 2 kb 7.1 ng/ul = 0.0035 equivalents/uL-
1 uL 10x T4 Ligase Buffer -
4.8 uL H20 -
0.5 uL T4 Ligase
Total=10 uL
leave at RT for 2 hours
Plated at 7:15, placed in 37C
Heat shock transformation of the psb1c3+hyb.ompa.aoxa (Scott)
10 սL BL21 cells + 5 սL of ligation reaction
(note, psb1c3 is choloamphenicol resistant!)
See Protocols Page for Heat Shock Transformation
picking colonies from psb1c3+hyb.ompa.aoxa a5 plates from 10.25.2010(Christina)
10/27/2010
Note: running low on miniprep spin columns and P2.
Miniprep of liquid cultures of psb1c3+hyb.ompa.aoxa A5 from 10/26
See Protocols Page for Plasmid DNA Purification with Mini Prep Kit
Picking colonies from psb1c3+hyb.ompa.aoxa a5 plates from 10.26.2010 (Christina, Scott)
Picked 25 colonies, started 9 am
Nanospec of minipreps of psb1c3+hyb.ompa.aoxa A5 (done today)
Digests
Digest of psb1c3+hyb.ompa.aoxa A5 from 10/27/2010 (minipreps from today)
we are digesting minipreps 1-7:
4.5 uL H20 -
2uL 10X EcoRI buffer -
10 uL hyb.ompa.aoxa.psb1a3 colony 5 miniprep (10.27.2010) (1-7)-
2 uL BSA (1ug/uL, to a final conc of .1mg/ml) -
0.75 uL SpeI
0.75 uL EcoRI
Total=20 ul total
put on heat block at 37c for 3 hours (start 10 am)
Digest of psb1c3+hyb.ompa.aoxa A5 from 10/27/2010 ( minipreps from today)
we are digesting minipreps 1-
5.25 uL H20
2uL 10X EcoRI buffer
10 uL hyb.ompa.aoxa.psb1a3 colony 5 miniprep (10.27.2010) (1-7)
2 uL BSA (1ug/uL, to a final conc of .1mg/ml)
0.75 uL EcoRI
Total=20 ul total
started 10:27 am
master mix
7.5*(5.25)=39.375 ul h20
7.5*(2)=15 ul buffer
7.5*(2)=15 ul bsa
7.5*(0.75)= 5.635 ul ecori
gels
wells 1-7=digest of psb1c3 minipreps 1-7 from morning of 10/27 using EcorI and SpeI
well 8=exactgene
wells 9-15=digest of psb1c3 minipreps 1-7 from morning of 10/27 using EcorI
(three versions of gel are presented below)
pcr using RFP-FR and Hyb-f+aoxa-R (Debika, Scott)
rxns
samples1-7 of psb1c3+hyb.ompa.aoxa
rxns:
1. rfp-FR (1-7 R)
2. Hyb-f Aoxa-R (8-14)
- 26.5 uL H2O-
- 10 uL Taq 5X Reaction buffer-
- 5 uL forward primer-
- 5 uL reverse primer-
- 1 uL dNTP 10 mM - (thawed & kept on ice)-
- 2 uL template DNA-
- 0.5 uL polymerase enzyme, Gotaq
Total Volume= 50 uL
1. master mix
7.5*(26.5)=198.75 ul h20-
7.5*(10)= 75 ul buffer-
7.5*(5)= 37.5 ul RFP-F-
7.5*(5)= 37.5 ul RFP-R-
7.5*(1)= 7.5ul dntp-
7.5*(0.5)= 3.75ul taq-
2. master mix
7.5*(26.5)=198.75 ul h20-
7.5*(10)= 75 ul buffer-
7.5*(5)= 37.5 ul Hyb-F-
7.5*(5)= 37.5 ul aoxa-R-
7.5*(1)= 7.5ul dntp-
7.5*(0.5)= 3.75ul taq
Colony PCR of transformations 5-10
2 reactions:
1. rfp-FR (1-7 R)
2. Hyb-f omp-R (8-14)
5 uL of cells
25.5 uL H2O
10 uL GOTAQ 5X Reaction buffer
5 uL forward primer
5 uL reverse primer
1 uL dNTP 10 mM - (thawed & kept on ice)
0.5 uL polymerase enzyme, TAQ
Total Volume= 50 uL
Chose miniprep 3 (from this morning)(44ng/ul) to send off)
part=BBa_K410000.
Tracking Number 796388355603
