Notebook:wlc

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8/21/12 RN NKG LF (Experiment 33)

Agenda for today: 1. Transform MLC2v and ANF plasmids into TOP10 cells. 2. Start vector double digest of pcDNA 3.1 + neo and pcDNA 3.1 + puro. 3. Pour more kana plates and amp plates 4. Autoclave microcentrifuge tubes.

Digests – 1 hour at 37 degrees Puro EcoR1 – buffer 4 Pme1 Neo Xbal1 – buffer 2 Bgl2

Transformations 5 microL of eluted plasmid to transform MLC2v and ANF plasmids each.

8/22/12 RN NKG LF (Exp 34)

Only one of the plates grew a colony from yesterday. ANF plate yielded no colonies. MLC2v plate yielded 1 colony.

Agenda for today: 1. Dilution plate the MLC2v colony from yesterday (33). 2. Transform ANF-eGFP plasmid and MLC2v-eCFP plasmid again into TOP10 bacteria through heat shock. 3. Work on wikipage and logo. Take pictures for wikipage and club.

Dilution plates Logan dilution plated the MLC2v colony, and it is growing up in the warm room. This colony has also been placed in a conical tube (LB + kana) and is also growing up in warm room on shaker.

Transformations The transformations of ANF and MLC2v plasmids are in the warm room, plated on amp and kana plates, respectively. We used 5 microL of each eluted plasmid, and all 250 microL was plated.

Assay We ran an assay to determine the amount of eluted plasmid in the microfuge tubes. (The original amount eluted from the filter paper). This was accomplished via the Qubit fluorometer.

(Exp 35)

We spoke with Dr. Balza about our plates from yesterday and the lack of DNA in the eluted plasmid (hoping just a very small amount, not missing altogether). He suggested that we use 10 microL of eluted plasmid and 20 microL of TOP10 cells for each transformation. So we are doing another transformation today.

(2) MLC2v eluted plasmid 10 microL TOP10 cells 20 microL

(2) ANF eluted plasmid 10 microL TOP10 cells 20 microL

We are heat shocking them as the transformation method.

Agenda for tomorrow: 1. Safety questions and abstract 2. Check plated TOP10 cells 3. Miniprep MLC2v and glycerol stock 4. Restriction digest on MLC2v 5. Gel for pcDNA3.1 + neo, puro, and MLC2v.

8/23/12 RN NKG LF (Exp 36)

Assay MLC2v plasmid from dilution plate (Qubit 25.4 microg/mL)

Dilution plating Dilution plating the MLC2v colonies grown up from 34 and 35. 6 plates total

From Exp 34 => 34-1, 34-2 From Exp 35 => 35-1,2,3,4

Labeled during lab work as 1-6

Gel Electrophoresis Gel of neo and puro digests

1 2 3 4 5 6 1kb (33) (34) Ladder puro neo

8/25/12 RN NKG SVA (Exp 39)

Agenda for today: 1. MLC2v double digest (of plasmids from exp 34, 35) 2. Gel Electrophoresis (of MLC2v) 3. Isolate neo and puro plasmids from yesterday’s gel 4. Cryogenically freeze MLC2v tubes.

8/26/12 RN NKG (Exp 40)

Double digest (39) gave ambiguous results. Looks like one enzyme did not work…we are redoing both enzymes separately for MLC2v. MLC2v 5 did not show anything on gel, so it is removed. MLC2v Digest (1-4, 6) Bgl2 Xbal Bgl2 and Xbal Control (plasmid with no enzymes added)

Neo Digest Bgl2 Xbal Bgl2 and Xbal Control

Puro Digest EcoR1 Pme1 EcoR1 and Pme1 Control

9/10/12 RN NKG 41

Double digest of MLC2v (1-4,6) and Neo plasmids Mfe1 Bgl2 Mfe1 and Bgl2 Control

Double digest of Puro plasmids Pme1 EcoR1 Pme1 and EcoR1 Control

Placed digests in 37 degree heat block.

(Exp 42)

Neomycin and Puromycin Resistant Feeder Layer The neomycin and puromycin resistant feeder layer project is starting today by Nick as well. Plated out STO cells for feeder layer transduction.

9/10/11 RN NKG (43)

Gel Electrophoresis A gel was run of all the digests performed in (41).

Gel Extraction We excised gel sections of the appropriate band size using UV light to visualize the DNA. Pre-weighed microfuge tubes were used to contain the gel sections. These are the lanes of gel from which we excised our DNA:

MLC2v (6), dd MLC2v (6), Bgl2 MLC2v (1), dd MLC2v (1), Mfe1 MLC2v (1), Bgl2 MLC2v (2), dd MLC2v (2), Bgl2 MLC2v (3), dd MLC2v (3), Mfe1 MLC2v (3), Bgl2 MLC2v (4), dd MLC2v (4), Mfe1 MLC2v (4), Bgl2 Neo, dd Neo, Mfe1 Neo, Bgl2 Puro, dd

9/12/12 NKG (45)

Followed Lab 3’s protocol to extract DNA from the agarose gel (Qiagex II Gel Extraction Kit (500)).

9/14/12 NKG RN (46)

Agenda for today: 1. Restriction digest for puro and neo 2. Gel electrophoresis 3. Gel extraction

Puro digests (4 each) – Pme1, EcoR1, dd, control Neo digests (4 each) – Bgl2, Mfe1, dd, control

Gel electrophoresis at 141v

9/15/12 LF (47)

Removed plasmids from ANF, Puro, and Neo

(48) RN NKG

QuantiT Assays to determine quantity of ANF, Puro, and Neo.

Plasmid Concentration (in sample, microg/mL) ANF 1A 15.2 ANF 1B 1.86 ANF 1C 5.71 ANF 2A out of range ANF 2B 1.03 ANF 2C out of range ANF T1 5.86 ANF T2 1.23

Neo 1.83 Puro 12.4

(49) RN

Gel Electrophoresis Gel of Neo and Puro restriction digest (double digest only). Gel showed no DNA, not even in the control.

(50) RN

Taking new colonies from ANF 1A, ANF 1B, ANF 1C, ANF 2A, ANF 2B, ANF 2C, ANF T1, ANF T2. (T-transfectant, 2-plate number, C-third colony from plate) Growing up in 7mL LB in warm room on shaker.

9/16/12 NKG RN (51)

Restriction double digest for ANF. We used Pme1 and EcoR1. Plasmids from (50).

(52)

Placed neo and puro bacteria in LB broth to grow up.

Gel Electrophoresis Gel is set for the ANF double digest.

15 microL of the digest/loading dye mix was placed into the wells so that we can extract the most DNA from the gel as possible, assuming it works.

Assay

Quantification of DNA using ANF plasmids.

Sample Concentration (microg/mL) 1 26.6 2 21.9 3 16.8 4 20.1 5 10.7 6 14.1 7 14.9 8 22.1

Low concentration of plasmid.

Gel was successful, bands of presumed ANF-eGFP constructs (from the double digest) have been cut out into microcentrifuge tubes for gel extraction.

Gel extraction

Extracting the DNA (plasmid) from the gel using the Qiaex II Gel Extraction Kit.

9/20/12 SVA RN LF NG (53)

Made agarose gel.

Purified neo and puro double digests from gel. Not sure if puro was correct size, but we cut it out anyway.

Also growing up new neo and puro bacteria for large amounts of plasmid.

Using puro plasmid from 9/17/12 to run single digests (Pme1, EcoRI). If the enzymes are negatively interacting with one another as we suspect, the single digests followed by purification and the other enzyme’s single digest will remedy this. Digest runs for 1 hour. Run in a gel to separate cut from uncut.

9/24/12 RN NKG (54)

New tubes of LB broth with neo plasmid and puro plasmid in e. coli. Purified out of bacteria Restriction digest for two hours.

Neo Mfe1 Bgl2 Double digest Control (plasmid alone)

Puro EcoRI Double digest Control

(running out of Pme1, so not running a single digest of Pme1)

Gel Extraction and Assay All neo and puro double digests as well as ANF from earlier show too low of concentrations to be useful.

NKG

Ran gel Removed correct bands DNA extraction from gel Ran concentrations for Neo, Puro, ANF, and MLC2v

9/26/12 LF NKG (55)

Combined all double digests of ANF 1-8 for the largest amount in one place. Combined all dd of MLC2v 1,2,3,4,6 Combined open vector Puro 1,2,3 into one open vector puro Combined open vector of Neo 1,2,3 into one open vector neo

9/28/12 LF NKG (56)

Ran gel electrophoresis, gel extraction, and qubit to determine concentration of DNA. Concentration is below detectable levels, so experiment has failed. Starting over with growing up plasmids in bacteria.

9/29/12 RN LF

Miniprep

Miniprep on all bacteria grown up to recover plasmid.

1-7 are MLC2v 8-15 are ANF

Double digest

controls run on 1-15 and puro and neo.

Pst1/EcoR1, buffer 3 used on all

Gel Electrophoresis

Open vectors from yesterday are still okay.

9/30/12 RN (58)

New LB from yesterday miniprepped.

10/1/12 RN (58)

Qubit for plasmids from 9/30/12. We will be sending those to MIT for them to sequence, since we had a difficult time with the ligation steps.

These two plasmids will be shipped to MIT:

number 4 MLC2v plasmid concentration 212 ng/mL part # BBa_K948000 number 10 ANF plasmid concentration 234 ng/mL part # BBa_K948000