Team:GeorgiaTech/WeekEight

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Plan for this week:

Weekend: PCR purify hybB, run gel for pSB1A3

Mon: Redo PCR RFP; digest RFP (O/N)
Tues: ligate hyB+RFP (O/N), Digest pSB1A3 (O/N)
Wed: PCR ; digest (1hr); PCR purify (digests of hyb+rfp and pSB1a3) at same time we run gel to check results; ligate O/N
Thurs: Tranformation of cells by hyb+RFP+psb1a3
Fri: check plates

9/20/2010

Goals:

Redo PCR of mRFP F,R; mRFP F2,R
Digest mRFP F,R; mRFP F2, R O/N (not possible until we get enzymes)
RUN GEL - 2 uL each - to check size - this step completed for all building blocks
PCR PURIFY leftovers - this step completed for all building blocks
NANOSPEC - record concentrations - this step completed for all building blocks
Transform novablue with mRFP that ryan gave us (labeled mRFP H) to create our own crytostock

Notes:

Ryan gave us a fresh stock of mRFP plasmid. We only get this one! It is precious like gold or diamonds!
Since the original mRFP has a Nde I site in the MCS, we are worried other products may run at the same size. To avoid this worry, we will use gel extraction to extract only our band of interest.

mRFP

in the plasmid PET15B
Use Novablue to create a cryostock. (transform, grow on plate, pick colony, grow in liquid media, take cryostock)

Protocols

9am

PCR Protocol for mRFP F,R; mRFP F2, R

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

0.5 uL polymerase enzyme, PHUSION

Total Volume= 50 uL

Ran PCR results (pre purification) on gel)

12pm

Heat shock transformation of the plasmids into our bacteria

10 սL Nova Blue cells + 5 սL of MRFP plasmid (from stock that Ryan gave us this morning).

See Protocols page for Heat Shock Transformation

Results

Gel picture of mRFP FR, F2R (Insert gel pic!)

The bands of both PCRs (pre digest) were between approximately around 700-800

Gel of mRFP FR and mRFP F2R

100bp ladder|mRFP FR|mRFP F2R|Control

PCR Purification-mRFP

See Protocols page for PCR Purification

Nanospec Results

mRFP, F: 137.2ng/uL

mRFP, F2: 132.3 ng/uL

For Future

Richard suggested getting two squirt bottles- 1 for water, 1 for bleach (to kill cells)
Check if transformation of Nova Blue with mRFP worked!

9/21/2010

Results from 9/20/2010

Transformation of mRFP in NB cells successful (both plates).

Starter Cultures

Make starter cultures from both mRFP:NB plates, allow to grow overnight.

3mL LB + 3uL 1000X CARB

put in 37C incubator with shaking overnight

Received shipment of SpeI (200 uL) and XmaI (50 uL) from Promega, put in -20C freezer (Gaucher Lab).

For 9/22/2010:

Begin digests 9am, do 3 hours insteadf of overnight, and ligation in evening
Create cryostocks from starter cultures grown on 9/21/2010.
Couldn’t start RE digest of RFP today- could not find RE’s. Will ask Megan Wednesday morning.

9/22/2010- (Mitesh, Scott, Gita)

Goals

Digest of RFP F,R
PCR purify the digest
Ligate hyBB FR, to RFP F,R

Notes: made a stock of 1 ug/ul BSA for easier RE digests

I am following the suggested RE protocol listed on the Promega literature that came with the RE’s

RE Digest Recipe for RFP-F,Rr

4.5 uL H20

2uL 10X Promega Buffer D

10 uL RFP-F,R ( from 9.20.2010, 137 ug/ul)

2 uL BSA (0ug/uL, to a final conc of .1mg/ml)

0.75 uL SpeI (edited changed from notI to speI )

0.75 uL NotI

Total=20 ul total

Run 3 hours in the heating blockin our lab (37c, put water in the heating blocks)

Start time is 10:35 am

End time should be 1:35 pm

Purification of RFP digest

See Protocols page for PCR Purfication

Nanospec

RFP Digest (purified)- 41 ng/uL

Ligation of RFP-FR to HybB-FR

using hybB from 9/18/2010 and RFP from 9/21/2010

Calculating equivalents:

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

hyBb-[26 ng/uL]/393 bp = 0.06 eq/uL

for linear ligations, use a 1:1 ratio of products

Ligation:

2 uL of RFP FR (SpeI, NotI digested; purified)

2 uL of HybB FR (EcoRI, NotI digested; purified)

1 uL 10x Ligase Buffer

4.5 uL H20

0.5 uL T4 Ligase

Total=10 uL

Leave RT for 1 hour

Start time: 3:20 pm

End time: 4:20 pm

PCR of hybB+Mrfp construct

3 uL of ligation reaction

25.5 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)

0.5 uL polymerase enzyme, PHUSION

Total Volume= 50 uL

Start: 5pm

End: Thursday

For Future

Take out pcr of mrfp+hybB
Run 2 uL on gel, along with just the RFP-FR and hybB-FR (1hr)
Digest the construct, along with the vector psb1a3 at the same time (3 hrs)
Ligate the construct to psb1a3 (1 hr)
Transform into e coli (1.5 hrs)

9.23.2010 (Scott, Rob, Gita)

Goals

Run PCR of RFP+hyBb on gel
Digest the construct, along with vecotr psb1a3
ligate the construct to psb1a3
transform into e. coli

Notes:

Predicted size of hyb+MRFP~1000bp

Protocols

Running PCR product on gel

The pcr was left overnight at 12 c, and I am running 4 uL on a gel
4 uL sample + 1 uL running dye

Notes:

I could not see any stained DNA, even the ladder. This means the EtBr degraded in the gels. Solution, suggested by Richard, is to keep a bottle of 1% Agarose/TBE solution at RT. This solution represents the first step of making a gel. Then whenever we need a gel, we start from the pre-made solution, add Etbr, and procede as normal. Start with fresh gels.
PCR products store in yellow box
Building blocks in blue rack

Making gel for PCR

1. Heat 30 mL of 1% agarose solution in microwave until agarose dissolves. Allow to cool. Make sure there are NO VAPORS before adding EtBr. EtBr is an intercalator. Don’t vaporize it, especially near your face!

2. Add 35 սL EtBr (edited from 45 uL, make 1000X)

3. Pour gel and allow to harden.

Results

Gel from 9.23.2010.

100 bp ladder| RFP-FR +HybB PCR|

Predicted Size- 1070 bp

Due to multiple bands, we decided to gel extract the construct RFP+hyBB. To do this, we borrowed a larger Gel try and apparatus from the Hammer Lab (they had a box of things they were not using). The larger gel is approximately 80 ml and is larger in area. The new gel will help make cutting the band easier.

Making gel for PCR (Hammer Lab Apparatus)

1. Heat 90 mL of 1% agarose solution in microwave until agarose dissolves. Allow to cool. Make sure there are NO VAPORS before adding EtBr. EtBr is an intercalator. Don’t vaporize it, especially near your face!

2. Add 90 սL EtBr (edited from 45 uL, make 1000X)

3. Pour gel and allow to harden.

(I did 100 mL, but found the actual volume to be around 80 to 90 mL)

Start: 4:10 pm

End: 5:10 pm

Gel Extraction Protocol

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).

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. To bind DNA, applied the sample to the QIAquick column, and centrifuged for 1 min.

7. Discarded flow-through and placed QIAquick column back in the same collection tube.

8. To wash, added 0.75 mL of Buffer PE to QIAquick column and centrifuged for 1 min.

9. Discarded the flow-through and centrifuged the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm).

10. Placeed QIAQuick column into a clean 1.5 mL microcentrifuge tube.

11. 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.

Gel of RFP-FR + HybB FR PCR product (Before Gel Extraction)

30 uL of PCR product|9.5 uL of 100 bp ladder

Band to be excised is the 1100 bp band (2nd bright band from bottom)

Gel of RFP-FR + HybB FR PCR product (After Gel Extraction)

30 uL of PCR product|9.5 uL of 100 bp ladder

For Future-

Friday-

nanospec the gel extracted hyb+RFP construct (in blue box)
Run 2 uL on gel, see if 1100 bp is confirmed
if so, continue with strategy.

9/24/2010Goals

nanospec the gel extracted hyb+RFP construct (in blue box)
Run 2 uL on gel, see if 1100 bp is confirmed
if so, digest the construct and vector psb1a3 (3 hrs); ligate (1hr), transform (1.5 hours)

Protocols

1 % Agarose Gel

I found that the small gel trays need only 25-30 mL of solution (less than 30)
I heated 30 mL of 1 % Agarose solution for approximately 45 seconds. I then added 30 uL of EtBr (use blue gloves) and poured into the prepared gel tray (tray inside of assembly, with combs). Cool for 1 hr. The gel will be done when there is a faint blue hue visible when looking at it.
Gel Start: 9:10 am
End 10:10 am

Nanospec of Gel Extract from RFP+HybB PCR

19.2 ng/uL

(inset gel pic from etbr lab)

Gel of Excised 1100 bp band of RFP-FR + HybB FR PCR product

4 uL of PCR product|9.5 uL of 100 bp ladder

Band to be excised is the 1100 bp band (2nd bright band from bottom)

RE Digest Recipe for HybB +RFP PCR (Mitesh did this today)

3.5 uL H20

3uL 10X Promega Buffer E

20uL HybB+RFP (excised 1100 bp badn from PCR) ( from 9.23.2010, 19 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

Run 3 hours in the heating blockin our lab (37c, put water in the heating blocks)

RE Digest Recipe for pSB1A3 (Debika did this today)

3.5 uL H20 (edit 9/24/2010- should be 8.5 uL)

5uL 10X Promega Buffer E or Multicore

10 uL pSB1A3 (9.16.2010, 100 ng/uL)

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

0.75 uL SpeI

0.75 uL EcoRI

Total=50 ul total

Run 3 hours in the heating block in our lab (37c, put water in the heating blocks)

PCR Purfiy the two digests (RFP+Hyb construct, pSB1A3)

Note- We have had the best results when eluting in 30 uL of H20

See Protocols page for PCR Purification

Nanospec

pSB1A3 Digest (purified) = 6.2 ng/uL

RFP+HYBB Digest (purified)= 4.4ng/uL

Ligation of pSB1A3 to [RFP-FR+ HybB-FR] (from 9.23.2010)

Calculating equivalents:

pSB1A3- [ 6.2 ng/uL]/ 2100 bp= 3x10^-3 eq/uL

hyBb+RFP-[ 4.4 ng/uL]/1100 bp = 4x10^-3 eq/uL

For plasmid to linear ligations, use a 1:2 ratio of vector:products if the products are purified (1:4 if not)

E.g. 1 equivalent of vector to 2 equivalent of linear product

Ligation Protocol:

3 uL of RFP + HYB (~ 2 to 3 uL)

2 uL of Plasmid pSB1A3 (~ 1/2x amount of linear product on eq/uL basis)

1 uL 10x Ligase Buffer

3.5 uL H20

0.5 uL T4 Ligase (enzyme-- keep in freezer-- add last of all)

Total=10 uL

Leave RT for 1 hour

Start: 5:23 pm

Transformation

Heat shock transformation of the plasmids into our bacteria

10 սL Nova Blue cells + 5 սL of Ligation Reaction

See Protocols page for Heat Shock Transformation

Notes for Future-

Aliquots of BL21 and Novablue are in our -70c freezer
Autoclave fresh pipette tips- there is confusion over which tips fit what pipettes- we need to calibrate to make sure our tips and pipettes are giving us the volumes we want

9/25/2010

Goals-

Begin digesting the building blocks necessary to create the rest of the constructs
Pick colonies from the pSB1A3+Hyb+RFP transformation and grow overnight

Nanospec results (From tubes dated 9/17/2010)

AOX1a-F,R = 99.3 ng/uL

AOX1a-F,R2 = 50.0 ng/uL

AOX1b-F,R = 48.5 ng/uL

AOX1b-F,R2 = 42.1 ng/uL

OmpA-F,R = 27.3 ng/uL

RFP-F2,R = 10.8 ng/uL

Hyb-F,R = 26.2 ng/uL

RFP-F,R = 8.8 ng/uL

RE Double Digest Recipe for RFP-F2,R

0 uL H20 (plenty in the RFP-F2,R tube, which is very dilute)

5 uL 10X Promega Buffer D

40 uL RFP-F2,R ( from 9.17.2010, 10.8 ng/ul)

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

0.75 uL SpeI

0.75 uL NdeI

Total=51.5 ul total

Start :4pm

End: 7 pm

RE Double Digest Recipe for OmpA F,R

1.5 uL H20

5 uL 10X Promega Multi-Core Buffer

37 uL OmpA-F,R ( from 9.17.2010, 27.3 ng/ul)

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

0.75 uL NotI

0.75 uL XmaI

Total=50 ul total

Went in at 2:55 pm

RE Double Digest Recipe for AOX1a F,R

12.5 uL H20

3 uL 10X Promega Buffer B

10 uL AOX1a-F,R ( from 9.17.2010, 99.3 ng/ul)

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

0.75 uL SpeI

0.75 uL XmaI

Total=30 ul total

Went in at 3:05 pm

RE Double Digest Recipe for AOX1a F,R2

2.5 uL H20

3 uL 10X Promega Multi Core Buffer

20 uL AOX1a-F,R2 ( from 9.17.2010, 50 ng/ul)

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

0.75 uL NdeI

0.75 uL XmaI

Total=30 ul total

Start 4 pm

RE Double Digest Recipe for AOX1b F,R

2.5 uL H20

3 uL 10X Promega Buffer B

20 uL AOX1b-F,R ( from 9.17.2010, 48.5 ng/ul)

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

0.75 uL SpeI

0.75 uL XmaI

Total=30 ul total

Went in at 3:10 pm

RE Double Digest Recipe for AOX1b F,R2

0 uL H20

3 uL 10X Promega Multi Core Buffer

22.5 uL AOX1b-F,R ( from 9.17.2010, 42.1 ng/ul)

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

0.75 uL NdeI

0.75 uL XmaI

Total=30 ul total

Start 4 pm

Run 3 hours in the heating block in our lab (37c, put water in the heating blocks)

Start time is 3, 4pm (two sets of reactions, noted in the individual descriptions)

End time should be 6,7 pm

Preparing liquid culture of pSB1A3+hybB+RFP

3 mL of LB + 3 uL of Carb into a culture tube. Picked 2 colonies from plates from 9.24.2010
O/N at 37c in our incubator-shaker

For Future-

PCR purify the digests
start first round of ligations
PCR the ligation products