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Latest revision as of 02:58, 28 October 2010

10/24/2010

 

Plated RFP expression studies begun:

 

 

 

 

 

 

 

 

 

 

10/25/2010

 

Goals

1. image hyb.ompa.rfp-f3r colony R11 plates to check for periplasmic expression (COMPLETED!)
2. obtain SpeI from Hammer Lab (COMPLETED)
3. digest A5 miniprep (10.23, in yellow rack) with EcoRI and SpeI (COMPLETED)
4. make more gels
5. this afternoon, gel extract the 1.5 kb isnert from A5 on a gel
6. 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)

1. 26.5 uL H2O-
2. 10 uL Taq 5X Reaction buffer-
3. 5 uL forward primer-
4. 5 uL reverse primer-
5. 1 uL dNTP 10 mM - (thawed & kept on ice)-
6. 2 uL template DNA-
7. 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