Team:GeorgiaTech/WeekTwelve

From 2010.igem.org

10/17/2010

Goals

1. transformations of NB of ligations done 10/16/2010

1. hyb.ompa.aox
2. hyb.ompa.aoxb
3. hyb.ompa.rfp-f3r
4. negative control

2. miniprep of aoxb colony b5
3. nanospec the minipreps done 10/16/2010 and today
4. check results of digests on gel

1. digests of hyb.ompa.aoxa/b colonies b1-3, a10 with EcoRI and Spe I

5. grow up a colony of psb1c3

Protocols

transformations of NB using ligations done 10/16/2010 (Christina)

1. hyb.ompa.aox
2. hyb.ompa.aoxb
3. hyb.ompa.rfp-f3r
4. negative control

10 ul of NB + 5 ul of ligation rxn

plate at 1 pm

gel of digests hyb.ompa.aoxa/b colonies b1-3, a10 with EcoRI and Spe I (Scott)

Note: problem with wells 2,3

order: ladder|b1|b2|b3|a10|b1|b2|ladder (exacto)

Colony aoxa A10 has the pattern we want: a 1 kb insert adn  2 kb bakbone

miniprep of colony b5 (Scott)

nanospec

hyb.ompa.aox.psb1a3 colony b5 (10.16.2010)=90 ng/uL

hyb.ompa.aox.psb1a3 colony b5 (10.17.2010)=101 ng/uL

Repeat pcrs to make hyb-Fr, RFP-FR, aox1b FR, aox1a FR

Rxns

Hyb-F and Hyb-R (note use 1 ul of template for hyb)

RFP-F and RFP-R

Aox1b-F and Aox1b-R

aox1a-F and aox1a-R

PCR Protocol

26.5 uL H2O -

10 uL PHUSION 5X Reaction buffer -

5 uL forward primer -

5 uL reverse primer -

1 uL dNTP 10 mM - (thawed & kept on ice)

2 uL template DNA (note: use 1 uL for hybB) -

0.5 uL polymerase enzyme, PHUSION -

Total Volume= 50 uL

grow up a starter colony of psb1c3 (Christina)

The colonies were pinkish, so Christina picked a colony so we can miniprep them tomorrow

Digest of colony B5 of hyb.ompa.aoxb.psb1a3 (from 10.16.2010)(Margo)

colony 5, Aox1b colony PCR from 10-16: Label on tube in freezer is 516

4.5  uL H20

2uL 10X EcoRI buffer

10 uL hyb.ompa.aoxb.psb1a3 colony 5 miniprep (10.16.2010)

2 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL SpeI

0.75 uL EcoRI

Total=20 ul total

colony 5, Aox1b colony PCR from 10-17: Label on tube in freezer is 517

4.5  uL H20

2uL 10X EcoRI buffer

10 uL hyb.ompa.aoxb.psb1a3 colony 5 miniprep (10.17.2010)

2 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL SpeI

0.75 uL EcoRI

Total=20 ul total

Into heat block at 1:51 p.m., remove at 4:51 p.m.

Making Gels

Started at 2:26 pm. Gels ready at 3:26 pm.

Gels:

1. 4 ul of the PCRs Christina did today

1. aoxa FR
2. aoxb FR
3. hyb FR
4. RFp FR

2. RE digest 516
3. RE digest 517

Gel started 5:00 pm.

Results:

10/18/2010

Goals

1. Send off for sequencing the minirep of colony a10

Colony PCR of hyb.ompa+aoxa+psb1a3,  hyb.ompa+aoxb+psb1a3,  hyb.ompa+RFP-F3R+psb1a3

2.5 uL of cells

11.75 uL H2O

5 uL GOTAQ 5X Reaction buffer

2.5 uL Hyb-F forward primer

2.5 uL Ompa-R reverse primer

.5 uL dNTP 10 mM - (thawed & kept on ice)

0.25 uL polymerase enzyme, TAQ

Total Volume= 25 uL

Picked four white colonies from each plate, added to 25uL water.

Run gel on colony PCRS from above.

Lanes were run in order (1-16), with a 1KB ladder middle band (there was only 2uL of this left, so the ladder is faint).

Gel electrophoresis of Top and Bottom bands from 17 OCT 2010

Ladder | Top | Bottom (yes, both are very faint)

Ladder | Top | Bottom

Retransformation of Ligations from 10-16

Repeated heat shock transformations of ligations from 10-16-10.

Used 5uL of ligation reaction + 10uL of NB cells.

Plated 100uL of each on each plate.

See Protocols page for Heat Shock Transformation

10/19/2010

Goals

1. reload colony pcrs from 10/18/2010 if need be (the ladder is faint, so if no interpretation is possible, then load again. otherwise no need to)  
2. Load digest the 2 digests of colony B5 hyb.ompa.aoxb.psb1a3 from 10.17.2010 (done)

1. if they have the expected insert, send off for sequencing

3. Load the pcrs from 10.17 of hyb-FR, aoxa-Fr, aoxb-FR, and rfp-F3R  on a gel (done)
4. pick (20) colonies of RFP-F3 from 10/18/2010 (done)

1. after the colony pcr, add 3ml of LB instead of 250 ul. This will prepare us for minipreps tomorrow.

5. Pick (20) colonies of aoxb (from 10.18/2010 plates) (done)

1. after the colony pcr, add 3ml of LB instead of 250 ul. This will prepare us for minipreps tomorrow.

6. try the ligation of hyb-rfp-psb1a3 , transform into NB or bl21
7. transform the “sequenced” colonies (a10) into bl21
8. miniprep of psb1C3 (done)
9. Send colony a10 for sequencing (Christina)(to be done)
10. grow up some more colony a10 from glycerol stock

Protocols

loading gel (done, Scott)

order:

exact ladder|aoxa-FR phusion pcr (10.17.2010)||aoxb-FR phusion pcr (10.17.2010)||hyb-FR phusion pcr (10.17.2010)||RFP-F3R phusion pcr (10.17.2010)|exact ladder| digest of miniprep of colony b5 (10.16 version) hyb.omp.aoxb.psb1a3 from 10.17.2010|digest of miniprep of colony b5 (10.17 version) hyb.omp.aoxb.psb1a3 from 10.17.2010

start 11:08 am

the pcrs appeared to work, but the digests do not contain the predicted band pattern.

Miniprep of psb1C3(Scott) (done)

prepare DNA to send off for sequencing.( Christina)(Scott)

In standard PCR tubes, prepare template DNA with just water and ONE primer (forward or reverse). The total volume should be 15 uL. The concentration of DNA in the sample should be adjusted to match the length of the template DNA; there is a chart in the Gaucher lab on the wall above the EtBr disposal bucket. For example, for a PCR product 1000 to 2000 bp in length, the concentration of the sample should be ~ 4 ng/uL.

Predicted size of hybB-ompA-aox1a: 1509 bp. Calculate concentration to be ~ 4 ng/uL

An email should be sent to Ryan with the following information:

Tube Label: actually written on the tube, in this form: RR01, RR02, etc.

DNA Name: this is something meaningful to us, like AOXa_construct_PSB1A3_vector_1

DNA Type: Purified PCR or Plasmid

DNA length: in bp

My primer: a name meaningful to us

Give the tubes to Ryan. Apparently they should not be frozen, just kept on ice.

Rxns: colony 10 hyb.ompa.aoxa miniprep from 10/15/2010

psb1a3-F

hybF

aox-F

aox-middle

colony PCRs (Rob, Christian, Scott)

use plates from 10/18/2010

use hyb-f and ompa-r

Reactions:

aoxa 1-20

aoxb 1-20

rfp-f3r 1-20

Colony pcr Master mix (batch of 70 amount in brackets)

11.75 uL H2O  (822.5 uL)-

5 uL GOTAQ 5X Reaction buffer  (350 uL)-

2.5 uL Hyb-F forward primer (175 uL)-

2.5 uL Ompa-R reverse primer (175 uL)-

.5 uL dNTP 10 mM - (thawed & kept on ice) (35 uL)-

0.25 uL polymerase enzyme, TAQ (17.5 uL)

2.5 uL of cells added to each respective PCR tube

Total Volume= 25 uL

Start: 3:31 pm

10/20/2010

goals

1. load rest of colony pcrs on gel from 10/19

1. load 5 ul

protocols

loading gels for colony pcrs (Scott)

expected fragments:

hyb-f and ompa-r approx 500 bp.

gel one (started 9:36 am)

order:

exact ladder|aoxa colony pcrs 1-6|ladder|aoxa colony pcrs 7-14|ladder

gel two

exact ladder|aoxa colony pcrs 15-20|colony pcr aoxb 1|ladder|two empty wells|ladder|aoxb colony pcrs 2-6|

gel three

order

exact ladder|aoxb colony pcrs 7-12 (from 10.19.2010)|ladder

gel four

order

exact ladder|aoxb colony pcrs 13-18 (from 10.19.2010)|ladder

(gel 3 and 4 are together below)

gel five

order

exact ladder|aoxb colony pcrs 19-20 (from 10.19.2010)|rfp-f3r colonies 1-4ladder

gel six

order

exact ladder|rfp-f3r colony pcrs 5-10(from 10.19.2010)|ladder

(gel 5 and 6 are below)

gel seven

order

exact ladder|rfp-f3r colony pcrs 10-20(from 10.19.2010)|ladder

RE Double Digest Recipe for pSB1C3 miniprep (from 10/19/2010) -- digest with speI and EcorI (Debika)

STARTED AT 2.54 PM

12.5 uL H20 -

3uL EcoRI Buffer -

10 uL pSB1C3 (10.8.2010, 56 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 2.54 PM

Predict a insert (1000 bp) an(?- this was already there from the last protocol that Scott copied for me, DM)

Things to do

1. load colonies rfp-f3r 10-20 on gel, visualize for 500 bp band (done Christina)

2. colony pcr using aoxa/b f-R from colonies on 10/19/2010 (Done Christina)

3. grow up good colonies in 5ml liq culture

colony pcr using aoxa/b f-R from colonies on 10/19/2010

Do 2nd pcr on colonies (from 10/19/2010)

aoxa: 3,5,6

aoxb: 15,18

rfp-f3: 1, 11, 12, 18, 19

Note: change the colony pcr program to have an extension time of 1.5 mins.

For aoxa (colonies 3, 5,6)

11.75 uL H2O  

5 uL GOTAQ 5X Reaction buffer  

2.5 uL Hyb F forward primer

2.5 uL Aoxa R reverse primer

.5 uL dNTP 10 mM - (thawed & kept on ice)

0.25 uL polymerase enzyme, TAQ

.25 uL of pcr reaction from 10-20-10 added to each respective PCR tube

Total Volume= 25 uL

For aoxb (colonies 15, 18)

11.75 uL H2O  

5 uL GOTAQ 5X Reaction buffer  

2.5 uL Hyb F forward primer

2.5 uL Aoxb R reverse primer

.5 uL dNTP 10 mM - (thawed & kept on ice)

0.25 uL polymerase enzyme, TAQ

.25 uL of pcr reaction from 10-20-10 added to each respective PCR tube

Total Volume= 25 uL

For RFP f3r (colonies 1, 11, 12, 18, 19)

11.75 uL H2O  

5 uL GOTAQ 5X Reaction buffer  

2.5 uL HybF forward primer

2.5 uL Rfp-r reverse primer

.5 uL dNTP 10 mM - (thawed & kept on ice)

0.25 uL polymerase enzyme, TAQ

.25 uL of pcr reaction from 10-20-10 added to each respective PCR tube

Total Volume= 25 uL

*NOTE - can use the PCR reaction (.25uL) from today on aoxa 3,5,6; aoxb 15, 18; rfp-f3 1 as template DNA.  (good point)

Ligation of RFP-FR,HybB-FR,to psb1a3 (Christina)

Calculating equivalents: 9C

rfp (from9.22.2010)

psb1a3 (10.15, top )

hyb (from 9.10, purified 10.20)

RFP- [41ng/uL]/678 bp= 0.06eq/uL

hyBb-[ 6  ng/uL]/393 bp = 0.0152 eq/uL

psb1a3-[ 9.4 ng/ul]/2000 bp= 0.0047 eq/uL

for linear ligations, use a 2:2:1 of insert:insert:vector  

0.5  uL of RFP FR

2 uL of HybB FR

3 ul psb1a3

1 uL 10x Ligase Buffer

3 uL H20

0.5 uL T4 Ligase

Total=10 uL

overnight 4c

for tomorrow

1. look on list for any overlaps and missed items
2. note: make glycerol stocks when doing minipreps