Team:GeorgiaTech/WeekTwelve

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10/17-10/23

10/17/2010

Goals

transformations of NB of ligations done 10/16/2010

hyb.ompa.aox
hyb.ompa.aoxb
hyb.ompa.rfp-f3r
negative control

miniprep of aoxb colony b5

See Protocols page for Plasmid DNA Purification with Mini Prep Kit

nanospec the minipreps done 10/16/2010 and today
check results of digests on gel

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

grow up a colony of psb1c3

Protocols

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

hyb.ompa.aox
hyb.ompa.aoxb
hyb.ompa.rfp-f3r
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)

See Protocols page for Plasmid DNA Purification with Mini Prep Kit

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:

4 ul of the PCRs Christina did today

aoxa FR
aoxb FR
hyb FR
RFp FR

RE digest 516
RE digest 517

Gel started 5:00 pm.

Results:

10/18/2010

Goals

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.

Plate	Microcentrifuge Tube Numbers
psB1A3-aox1A (negative control, 10/17)	1,2,3,4
psB1A3-aox1B-hybB-ompA (10/17)	5,6,7,8
psB1A3-aox1A-hybB-ompA (10/17)	9,10,11,12
psB1A3-hybB-ompA-RFP,F3R (10/17)	13,14,15,16

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

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)
Load digest the 2 digests of colony B5 hyb.ompa.aoxb.psb1a3 from 10.17.2010 (done)

if they have the expected insert, send off for sequencing

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

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

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

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

try the ligation of hyb-rfp-psb1a3 , transform into NB or bl21
transform the “sequenced” colonies (a10) into bl21
miniprep of psb1C3 (done)
Send colony a10 for sequencing (Christina)(to be done)
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)

See Protocols page for Plasmid DNA Purification with Mini Prep Kit

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

load rest of colony pcrs on gel from 10/19

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

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

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

10/21/2010

goals

glycerol stocks of liquid cultures (done)
repeat pcr from 10/20 checking the “good” colonies; the gel showed no predicted bands, but that may be due to pcr errors, elongation time etc. Try using aoxa/b f-r only, or try troubleshooting why the pcr did not make the 1.5 kb band
miniprep liq cultures of confirmed colonies (Scott)(done)

nanospec’ed

send ones that are confirmed for sequencing
digest the three inserts from 3 good colonies and ligate to psb1c3

make sure to pcr purify the digest from yesrerday

transform bl21 using hyb-rfp ligation
transform bl21 using minipreps of good colonies
check list for anything else!

protocols

glycerol stocks of liq cultures(Christian)

colonies:

aoxa: 3,5,6

aoxb: 15,18

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

loading gel to check pcrs yesterday (Scott)

rxns:

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

aoxa using hyb-f and aoxa-r: 3,5,6

aoxb using hyb-f and aoxb-r: 15,18

rfp-f3 using hyb-f and rfp-r: 1, 11, 12, 18, 19 (used hyb-f and rfp-r)

order

version 2 of above gel:

RE Double Digest Recipe miniprep (from 10/21/2010) -- digest with speI and EcoRI

12.5 uL H20 -

3uL EcoRI Buffer -

5 uL miniprep -

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

0.75 uL SpeI

0.75 uL EcoRI

Total=30 ul total

3 hour digest

started 1pm

predicted sizes:

2kb (backbone)

hyb.ompa./aoxa/b insert approx 1kb

pcr using aoxa/b FR (done)

rxns

aoxa using aoxa-f and aoxa-r: 3,5,6-

aoxb using aoxb-f and aoxb-r: 15,18-

rfp-f3 using rfp-f and rfp-r: 1, 11, 12, 18, 19- (used hyb-f and rfp-r)

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

started 1:30pm

prepare DNA to send off for sequencing.( Margo)

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 (check sheet)

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 aox5(a5) , aoxb 18(18b), rfp-f3r colony 1 (1r)

psb1a3-F

hybF

aox-F

aox-middle

RR01 = Aox1a colony A5, with psb1a3 forward primer

RR02 = Aox1a colony A5, with AOX1a forward primer

RR03 = Aox1a colony A5, with AOX1a middle forward primer

RR04 = Aox1a colony B18, with psb1a3 forward primer

RR05 = Aox1a colony B18, with AOX1b forward primer

RR06 = Aox1a colony B18, with AOX1b middle forward primer

RR07 = RFP colony R12, with psb1a3 forward primer

RR08 = RFP colony R12, with RFP3 forward primer

RR01 AOX1a_colonyA5_psb1a3_primer 1500 bp psb1A3_forward

RR02 AOX1a_colonyA5_aox1af_primer 1500 bp aox1a_forward

RR03 AOX1a_colonyA5_aox1amid_primer 1500 bp aox1amid_forward

RR04 AOX1b_colonyB18_psb1a3_primer 1500 bp psb1A3_forward

RR05 AOX1b_colonyB18_aox1bf_primer 1500 bp aox1b_forward

RR06 AOX1b_colonyB18_aox1bmid_primer 1500 bp aox1bmid_forward

RR07 RFP_colonyR12_psb1a3_primer 1200 bp psb1A3_forward

RR08 RFP_colonyR12_RFP3_primer 1200 bp RFP3_forward

aoxa using aoxa-f and aoxa-r: 3,5,6-

aoxb using aoxb-f and aoxb-r: 15,18-

rfp-f3 using rfp-f and rfp-r: 1, 11, 12, 18, 19- (used hyb-f and rfp-r)

loading gel

gel 1 and 2 are of digests using ecoRI and SpeO

Gel 1(left)

ladder|A3|A5|A6|B15|B18|empty|ladder

Gel 2(right)

ladder|R1|R11|R12|R18|R19|empty|ladder

gel 3

PCR from 10.21.2010 using aoxa/b-FR or rfp-FR (for Rfp-f3r constructs)

of colonies from hyb.ompa.aoxa/b or rfp-f3r colonies

order

transformations into bl21: (Christina, Christian)

aoxa: 3,5,6-

aoxb: 15,18-

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

transformation of hyb-rfp-psb1a3 into bl21(Margo, Christina)

10 սL Nova Blue cells + 10 սL of ligation rxn

See Protocols page for Heat Shock Transformation

to do(in addition to list)

make more lb +carb plates

autoclave eppendorfs, toothpicks, tips

miniprep liq cultures of confirmed colonies (Margo)(done)

See Protocols page for Plasmid DNA Purification with Mini Prep Kit

nanospec(margo done)

TAKE OFF DOUBLE DIGESTS AT 4 PM (Heat block) (digests done today)

we put these back on to go overnight

10/22/2010

goals

take pictures of the two plates labeled “hyb.rfp.psb1a3” before putting them into the 4c to track changes in colony color (Margo, Done)
run digests from yesterday on gel - done (Christian, Margo)
make lb+carb plates - done (Margo, Scott)
using the transformations of the aoxa/b and rfp-f3r colonies as templates, resmear additional plates so that we can have distinct colonies as opposed to the lawns we have now - done (Margo)

Protocols

Making LB media (Margo, Scott)

To make 0.75 L LB (Lysogeny broth) medium:

3.75 g Yeast extract

7.5g Tryptone

3.75g NaCl

To make solid plates, follow the above recipe and add 16g agar per 1L (12 for .75 l)

Prepared 800 mL of LB media (50 ml extra)

750 ml for plates and 250ml liquid

Autoclave, leaving lids slightly loosened.

Let cool in water bath at 55C. (started 12 pm)

Add antibiotic (800uL carbenicillin) to 800 mL LB media used for plates.

For the agar plates, pipetted 25mL into each plate, allow to hardened on benchtop. Refrigerated and ready to go..

Note for others:

For resmearing the plates of the transformation done 10.21.2010 (aox 3, 5a, 6a, aox b15 and b18, rfp f3 1, 11, 12, 19). Make sure to save ALL plates when done and put the ones from 10.21.2010 back into the 4c fridge! We are tracking the progress of red colonies on the rfp-f3 paltes

Made liquid cultures of colonies A3, A5, and R11 from cryostocks; in incubator ~ 1:15 p.m. (Margo)

Digest Gel

(100bp) Ladder|1R|11R|12R|18R|19R|Ladder|A3|A5|A6|Ladder|B15|B18

Made liquid cultures of colonies of hyBb-RFP-psB1a3 in BL21. I picked 6 colonies from plate 1. #6 was already red by the time I started the culture, the rest were pinkish. I also picked 7 colonies from plate 2. #12 and #13 were already red, and the rest were pinkish by the time I started the culture.

The 13 tubes are in the 37 degrees incubator/ shaker in the other room.

How to prepares samples for microscopy (Look at table below)

1. In cold, growing for a couple of days.

Get a fresh plate...liquid of streaking of a colony.

2 or several plates for cold temperature. 1 will be left at cold until the last minute. the other one should be moved to warm a couple hours before we go to the scope.

2 other plates... put them in 37 and grow them overnight... (saturday night let them grow), sunday transfer them to the cold, one of these will stay in the cold until the last minute, and one will be moved to warm a couple hours before we image.

another plate... at 37 the whole time.. never goes to cold. - do this on sunday preferably.

Once we have new plates that have been streaked with colonies from liquid culture on Saturday night, prepare the following samples: (I explaine this to Scott. If he’s not here, call him or me!)

Plate	When you have to move plates to new temperature	Conditions
1		Leave in fridge (4 degrees) until we image
2	Sunday night	Leave in 4 degrees until the last person leaves on Sunday night, put in 37 degrees incubator.
3	Monday morning	Leave in 4 degrees until Monday morning at 7 am, put in 37 degrees

4	Monday morning	Leave in 4 degrees until Monday morning at 8 am, put in 37 degrees
5	sunday day time	Leave in 37 degrees overnight (Saturday), move to cold on Sunday (daytime). Leave in cold until we image
6	Sunday day time, sunday night	Leave in 37 degrees overnight (Saturday), move to cold on Sunday (daytime). Move back to 37 sunday night before you leave.
7	Sunday night, monday morning	Leave in 37 degrees overnight (Saturday), move to cold on Sunday (daytime). Move to 37 at 7 am on Monday morning
8	Sunday night, monday morning	Leave in 37 degrees overnight (Saturday), move to cold on Sunday (daytime). Move to 37 at 8 am on Monday morning
9	never	Leave in 37 the whole time

10/23/2010

goals (look at list in lab)

protocols

Digest of colony A5 of hyb.ompa.aoxA.psb1a3 (from 10.22.2010)(Margo)
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

Note: we need SpeI or a equivalent enzyme with same cutting site and overhang! We couldn’t start the digest today!

observations

on the hyb-rfp plates from 10.22, we saw some colonies that were slightly pink that were not pink yesterday; they are circled and dated with today’s date.

heat characterization

Making 3 l of lb and 1 l of lb+agar (Margo, Christian)
we plated each of the 9 conditions (above table) using colony R11 liquid cultures from 10.22.2010

plates are in appropriate 4c or incubator conditions

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