20240408-20240502: Molecular Biology Experiments

Molecular biology experiments
Overview	Specific content
Resuscitation strains	Resuscitation of pet28a-BMP-4-DH5, pet28a-VEGF DH5
Extract plasmid, prepare target gene fragment by PCR, cut pET28a plasmid by nco1&xho1, connect	The gel concentration after enzyme digestion of plasmid is extremely low
PCR obtained FTD, pet28a backbone, BMP2 sequence, and electrophoresis verification	The PCR template for the fragment used for seamless cloning is FTD and BMP-4 produced by the primers P above.
PCR was used to prepare FTD and BMP-4 templates, and PCR was used again to prepare SM FTD and BMP-4 sequences.	BMP-4 SMBMP-4 M (from bottom to top: 100 250 500) FTDD SMFTD
PCR preparation of pet28a template	Gradient PCR determines the annealing temperature of vector preparation. As the temperature rises, large fragments increase and small fragments decrease.
Preparation of pet28a backbone, SM-FTD, SM-BMP-4, gel excision recovery/PCR product purification and seamless cloning
There are a lot of colonies on the plates for seamless cloning BMP-4 and FTD, but there are also colonies on the negative control plate, but they are very few in comparison.
Colony PCR verification	1~7: BMP-4 No. 1~7; 8~12: FTD No. 1~5; 13: marker; 14~15: FTD No. 6~7; 16~19: seamless cloning negative pair; 20~21: PCR negative pair (I don’t know why the 16~19 seamless cloning negative pair also exploded, but it can be obtained by sequencing the connection product obtained earlier, so it’s not a big problem)
Top 10 strains of B-pet28a and FTD-pet28a were stored and plasmids were extracted	Transform FTD-pet28a and BMP2-pet28a
into six expression competent cells (BL21 rosetta shuffleT7, B&k12 AE, BL21Plyss)	FTD's have all grown out, B's BL21plyss and rosetta have not grown out, waiting to be re-done
Re-transfer of BL21plyss and rosetta to BMP-4	All the re-transplanted ones have grown out, pick a single colony
Colony PCR	T7 primer amplified BMP-4 fragment: 531bp; T7 primer amplified FTD fragment: 882bp; T7 primer amplified sfGFP fragment: 880bp (marker: Sangon Biotech 2K) From left to right in the above picture: AE, BL21, R, P, ShuffleT7B, ShuffleT7K12, BL21 is the FTD gene, and the rest are BMP-4 genes T7 primer amplified BMP-4 fragment: 531bp; T7 primer amplified FTD fragment: 882bp (marker: Sangon Biotech 2K) ShuffleT7K12
Fermentation test to see who can express soluble protein better	Collect bacteria, crush

20240520-20240615: Protein Fermentation and Purification

The cultured bacteria were collected, ultrasonically broken one by one, and centrifuged at 17000xg. The samples were whole bacteria, supernatant, and precipitate.

Solarbio marker

The lanes on the right of the marker are divided into four groups, one of which is BL21, AE, ORIGAMI, and SHUFFLEYT7. The four groups are whole bacteria, supernatant, and precipitate. Except for the supernatant group of shuffleT7, which was not diluted, the samples in the other 11 lanes were diluted twice.

I found that BL21 could not express and the yield of shuffleT7 was too low. Both rosetta and origami had proteins in the supernatant, but considering the growth rate, I chose AE because rosetta grows faster.

Concentration (ug/ml)

275.9038 FTDBMP4 concentrate

10.65392 50KDA concentrate↑

20240701-20240910: Cell Characterization Experiments

See the figure for the plating design and related calculations

The lyophilized powder was dissolved in 1 mL of tris-hcl (pH 8.0)

1. Biocompatibility:

Live and dead fluorescence staining d1

Cell proliferation (such as CCK8) d1d3d7

2. Cell attachment:

Cytoskeleton and cell nucleus immunofluorescence staining d1

Observe cell morphology using SEM after gradient dehydration of cells d1

3. Osteogenic differentiation:

Alkaline phosphatase expression images d1d7d14

Alizarin red staining d1d7d14

Cell culture, digestion and cryopreservation

(1) Growth medium: α-MEM +10%FBS+1%PS

Osteogenic induction medium (chemical): Growth medium plus 10 mM sodium glycerophosphate, 60 mg/L ascorbic acid, dexamethasone 100 nM (valid for 1 month, see next page for specific amounts)

(2) Cell digestion: remove the culture medium, add 5-10ml PBS to wash once, add 3ml pancreatin to the T75 bottle, digest in the incubator for 3min, stop digestion with 5ml growth medium, and centrifuge at 1000rpm for 5min.

(3) Cryopreservation solution preparation DMSO:FBS:culture medium = 1:2:7, cell volume 1 x106/ml cryopreservation solution,

First, mix 2 parts of serum, 2 parts of culture medium and 1 part of DMSO evenly, and then slowly add the mixture to the cell-culture medium system to achieve a slow increase in DMSO concentration to reduce DMSO damage to cells.

Use gradient cryopreservation boxes for freezing. Gradient cryopreservation boxes need to be restored to room temperature before use. After being placed in the cryopreservation box for at least 7 hours, they can be transferred to liquid nitrogen for storage.

2. Recommended cell inoculation volume

Cell culture plate specifications Cell inoculation volume (/well) Culture medium volume Related experiments

96-well 1x104 200 μl CCK 8. Live-death staining

24-well 5x104 1ml Differentiation (staining)

6-well 2x105 1.5ml-2ml Differentiation (mRNA)

T75 bottle 1 x106 (rats grow 3x106; humans grow 1.8 x106) 12-15ml Cell culture

White cell culture plate packaging paper is an adherent culture plate, and green is an anti-adhesive plate!!

3. Osteogenesis-related detection

Observe trend changes (first experiment), set time points for 1, 4, 7, 14, 21, and 28 days for detection

If there is no need to observe trends and mature experimental systems, 14 days can be selected as the detection time point (except Alizarin Red)

Detection Recommended reagent brand Time point (optional)

ALP quantitative detection Beyotime P0321S 14 days<

Calcium content detection Nanjing Jiancheng C004-2-1 21 days/28 days

dsDNA (normalized Yisheng 12641ES03 Same as ALP/calcium content detection

ALP staining Beyotime C3206 14 days

Alizarin red staining Solarbio 21 days/28 days

Immunofluorescence RUN-2, BMP 2, Collagen I, Abcam 14 days

OCN, OPN

50mL osteogenic culture medium requires:

108mg sodium β-glycerophosphate

3mg ascorbic acid

1.96ug dexamethasone